Method of altering the immundominance hierarchy of HIV gag by DNA vaccine expressing conserved regions

ABSTRACT

The invention provides methods and compositions for eliciting broad immune responses. The methods employ nucleic acid vaccines that encodes highly conserved elements from a virus.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage of International Application No.PCT/US2013/028932, filed Mar. 4, 2013, and which claims the benefit ofU.S. provisional application No. 61/606,265 filed Mar. 2, 2012, which isherein incorporated by reference for all purposes.

BACKGROUND OF THE INVENTION

HIV-1 strains are highly variable and this diversity provides a majorchallenge for vaccine design. A candidate vaccine should provideprotection against most clades of HIV. To address this problem,approaches to maximizing immunological strength and breadth are beingexplored, including strategies that use consensus, center-of-tree orancestral sequences, multiple strains or mosaic immunogens, immunogensconsisting of known epitopes from the database, and chimeric, moleculesexpressing a selection of the most conserved epitopes from differentclades of HIV [1-17, 56, 96 ].

REFERENCE TO SEQUENCE LISTING

This application includes a Sequence Listing as a text file named“77867-913960-606100US-SEQLIST.txt” created Jul. 30, 2014, andcontaining 104,770 bytes. The material contained in this text file isincorporated by reference in its entirety for all purposes.

In addition to sequence diversity, the presence of potentialimmunodominant epitopes provides another hurdle in the development ofeffective HIV vaccines. Accumulating evidence indicates thatimmunodominant epitopes exist, and that they may constitute animpediment for the production of effective universal HIV vaccines[18-31], as subdominant epitopes within HIV proteins have generally beenassociated with virologic control [19,22]. The use of any geneencompassing a complete protein as immunogen contains variable as wellas conserved regions. Since variable sequences can mutate to escapeimmune responses while retaining function, and can containimmunodominant T cell epitopes, we argue that variable segments shouldbe excluded from the design [32]. Our vaccine approach thus focuses onthe induction of immune responses to nearly invariable proteomesegments, many of which should be essential for the function of thevirus, and the prevention of responses against variable segments andpotentially immunodominant “decoy” epitopes [32-34].

The conserved element approach is supported by the followingobservations: (i) Viral proteins recover ancestral amino acid (AA)states when transmitted to a new host [35], and in the absence of thespecific immune responses found in the previous host, they can recover amore fit state [36-38]; (ii) changes in conserved AA of viral proteinscan destroy or significantly weaken HIV, indicating a critical role invirus biology [39-42]; (iii) CTL responses against specific viralproteins (e.g., Gag) are associated with relative control of viremia[43-50], and in the case of controllers and long-term non-progressors,high avidity CTLs targeting conserved regions have been identified[34,51]; (iv) immunodominance of some epitopes can obscure or preventreactivity against other, potentially protective epitopes [52]; (v) someAA segments in viral proteins are conserved throughout a given HIV-1subtype, the entire group M, and, in some instances, in HIV-2 and SIV[32,53]. Together, these considerations predicted that an HIV vaccinethat does not contain variable epitopes, and thus lacks potentiallyimmunodominant decoy epitopes, but instead consists of strictly,conserved proteome elements is better fit to induce immune responsesable to prevent virus acquisition or virus propagation [32,53]. Theconserved elements used in our work differ from those used by others[11,12,16,17,54-56] that were selected using different criteria, as wehave focused on both conservation and associations of particularsequences with immune control.

Previous work has been performed using Gag as a prototype vaccine,because Gag-specific T cell responses were found to correlate withcontrol of viremia in clade B and C infected individuals [43,48-50].Seven highly conserved elements (CE) were identified in HIV-1 p24^(gag)[32,34] (see also FIG. 1A). Indeed, a cross-sectional ex vivo studyshowed broad recognition of several CE in the context of wide HLAdiversity and identified T cell responses of high functional avidity andbroad variant reactivity [34], predominantly in controller individuals,suggesting an association between these T-cell responses and HIVcontrol.

The present invention address the need for an improved protocol forinducing an immune response by providing a strategy based on employingDNA constructs encoding conserve elements in conjunction with constructsencoding the substantially full-length protein from which the conservedelement vaccine is derived.

BRIEF SUMMARY OF THE INVENTION

The immunogenic regimens of the present invention focus on immuneresponses to proteome segments important to the function of a protein,e.g., a viral protein such as a lentiviral gag protein, and precluderesponses against segments that absorb much of the host immune response,but which can mutate to escape immune responses while retaining function(often referred to in the art as “immunodominant decoys”) (32). Aconserved element vaccine (CEvac) administered in accordance with theinvention has properties of a universal vaccine against a virus, such asa lentivirus, e.g., HIV, and is able to induce immune responses to mostor all circulating strains. In one embodiment, the invention provides amethod of generating an immune response where the method comprisesadministering a p24^(gag) DNA vaccine that expresses conserved elements,e.g., from 3 to 7 conserved elements (CE), of HIV-1 p24^(gag) andexcludes potential immunodominant Of variable regions acting aspotential decoy epitopes (32); followed by administration of a DNAvaccine encoding a full-length gag, e.g., p55^(gag).

In some embodiments, the invention provides a method of inducing broadimmune response including those direct to the highly conserved elements,the method comprising vaccinating with CEvac DNA and gag DNA eithersequentially or by co-immunization where responses are not produced uponvaccination with full-length p55gag immunogen. This vaccination approachovercomes the problem of diversity by generating cross-cladegag-specific immune responses and broadens the p55gag induced T cell andhumoral immunity. In some embodiments, the vaccines are delivered as DNAvaccines, e.g., plasmid DNA vaccines. In some embodiments, the vaccinesare delivered as adenovirus vaccines or vaccinia virus vaccine, or usinganother virus vector-based vaccination strategy.

In some embodiments, the invention provides vaccine compositionscomprising a nucleic acid encoding six or seven highly conservedelements from p24gag. In some embodiments, the elements encoded by thenucleic acid are arranged collinearly. In some embodiments, the sevenelements are separated by alanine linkers for efficient proteolyticcleavage. In some embodiments, DNA vectors are engineered to express theCore proteins (conserved element polypeptides comprising multipleconserved elements) only, to express secreted Core proteins having theN-terminal GM-CSF signal peptide (SPCore) or to express as a core fusionto the monocyte chemoattractant protein 3 (MCP3) chemokine to stabilizethe protein expression and enhance secretion of the proteins, or to thelysosomal associated membrane protein 1 (LAMP-1) to direct the proteinsto the lysosomal compartment including access to the MHC class IIpathway.

In some embodiments, a method of the invention comprises administering anucleic acid encoding a conserved element from a protein, e.g., a viralprotein such as Gag, and a nucleic acid encoding a naturally occurringvariant of the conserved element, where the variant differs from theconserved element by 1 amino acid, e.g., where the conserved element is8 amino acids in length; or has at least 50%, typically at least 90% orgreater sequence identity to the conserved element. In some embodiments,the variant may different from the conserved element by 1, 2, or 3 aminoacids. The nucleic acids encoding the conserved element and variantconserved element may be present on the same vector or encoded bydifferent vectors. In some embodiments, the conserved element is fromHIV gag. In some embodiments where a nucleic acid encoding a conservedelement and a nucleic acid encoding at least one variant conservedelement are administered, the conserved element and variant conservedelement sequences together account for at least 80%, or at least 90%,typically at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ofnaturally occurring variants of the protein. In some embodiments,conserved elements immunogens are constructed based on “regional focus”,which will account for >80% of clade C HIV-1 or clade B HIV-1. Such anapproach, for example, provides more specific vaccines.

The invention thus provides a methods of generating a broad immuneresponse, the method comprising administering a nucleic acid encoding atleast three conserved elements, typically at least 4, 5, or 6, or moreconserved elements, from a protein of interest, to an individual andadministering a nucleic acid encoding the full-length protein to theindividual. In some embodiments the conserved element is from a highlydiverse viral proteins, e.g., an HIV protein such as HIV gag or env.

In one aspect, the invention provides a method of inducing an immuneresponse in a subject, the method comprising administering a first Gagconserved element nucleic acid that encodes a first Gag conservedelement polypeptide (“CE1 polypeptide”) that comprises six conservedelements from Gag to the subject, wherein the conserved elements arefrom different regions of Gag, and further, wherein each conservedelement is at least 12 amino acids in length, but less than 30 aminoacids in length and the conserved elements are not contiguous; andadministering a nucleic acid encoding a full-length Gag protein. In someembodiments, the conserved elements are from HIV-1 p24gag. In someembodiments, the first conserved element polypeptide comprises at leastone conserved element that has an amino acid sequence set forth in SEQID NOS:1-7, 32, or 33. In some embodiments, the first conserved elementpolypeptide comprises at least two, three, four five, or six conservedelements that have an amino acid sequence set forth in SEQ ID NOS:1-7,32, or 33. In one embodiment, the first conserved element polypeptidecomprises conserved elements that each have a sequence set forth in SEQID NOS:1-7; or the first conserved element polypeptide comprisesconserved elements that each have a sequence set forth in SEQ IDNOS:3-6, 32, and 33.

In some embodiments, the methods of the invention further compriseadministering a second Gag conserved element nucleic acid that encodes asecond Gag conserved element polypeptide (“CE2 polypeptide”) thatcomprises at least one variant of a conserved element contained in thefirst Gag conserved element polypeptide, wherein the variant in the 2ndpolypeptide differs from the variant in the first polypeptide by 1, 2,or 3 amino acids. Typically, the variant conserved element differs fromthe conserved element by only 1 amino acid. In some embodiment, theconserved element and variant conserved element each have a sequence setforth in FIG. 1; or FIG. 13. In one embodiment, the first conservedelement polypeptide comprises the sequence of SEQ ID NO:15 and thesecond Gag conserved element polypeptide comprises the sequence of SEQID NO:16. In some embodiments the first conserved element polypeptidecomprises the sequence of p24CE1c and the second conserved elementcomprises the sequence of p24CE2c as shown in FIG. 13. In someembodiments the first conserved element polypeptide comprises thesequence of p24CE1d and the second conserved element comprises thesequence of p24CE2d as shown in FIG. 13. The first and second Gagconserved element nucleic acids can be administered sequentially orconcurrently. In some embodiments, one or more of the conserved elementpolypeptides comprise a signal peptide, such as GM-CSF or MCP-3. In someembodiments, one or More, of the conserved element polypeptides comprisea sequence that targets the protein for degradation, e.g., a LAMPsequence. In some embodiments, the first and second nucleic acid Gagconserved element polypeptides are encoded by the same vector. In someembodiments, the first and second nucleic acid Gag conserved elementpolypeptides are encoded by different vectors. The nucleic acidsencoding the first and second conserved element polypeptides may beadministered multiple times. In some embodiments, the nucleic acidencoding the full-length Gag protein is administered after a nucleicacid encoding a conserved element polypeptide, e.g, at least 2 weeks or4 weeks, or longer after a nucleic acid encoding a conserved elementpolypeptide.

In a further aspect, the invention provides a method of inducing animmune response to an HIV gag protein, the method comprising

-   (a) administering:-   (i) a nucleic acid encoding a polypeptide comprising SEQ ID NO:15    and a nucleic acid encoding a polypeptide comprising SEQ ID NO:16 to    a subject; or-   (ii) a nucleic acid encoding a polypeptide comprising p24CE1d as    shown in FIG. 13 and a nucleic acid encoding a polypeptide    comprising p24CE2d as shown in FIG. 13 to the subject; or-   (iii) a nucleic acid encoding a polypeptide comprising p24CE1d as    shown in FIG. 13 and a nucleic acid encoding a polypeptide    comprising p24CE2d as shown in FIG. 13 to the subject; and-   (b) administering a nucleic acid encoding p55gag.

In some embodiments, the nucleic acid pairs set forth in (i), (ii), or(iii) of step (a) are encoded by the same vector. In some embodiments,the polypeptides are fused to a GM-CSF signal peptide. In someembodiments, the nucleic acid encoding p55 gag is administered at leasttwo weeks after step (a).

In a further aspect, the invention provides a method of inducing animmune response to an HIV gag protein, the method comprisingadministering at least one nucleic acid encoding a conserved elementpolypeptide comprising a sequence set forth in SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:21, SE ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SE IDNO:29, SEQ ID NO:31, SEQ ID NO:35, SEQ ID NO:37, ear SEQ ID NO:39 to thepatient; and administering a nucleic acid encoding a full-length gagprotein. In some embodiments, the full-length gag protein isadministered at least 2 weeks after administering the nucleic acidencoding the conserved element polypeptide.

In a further aspect, the invention provides a method of inducing animmune response to a protein of interest, the method comprisingadministering a nucleic acid encoding a conserved element polypeptide,wherein the conserved elements are from the protein of interest and thepolypeptide comprises at least three conserved elements, each of lessthan 30 amino acids in length where the conserved elements are joined bylinkers; followed by administering a nucleic acid encoding thefull-length protein, wherein the nucleic acid encoding the full-lengthprotein is administered at least two weeks after the nucleic acidencoding the conserved element polypeptide.

In some embodiments the nucleic acid constructs encoding the conservedelement polypeptides and full-length Gag polypeptide are administeredintramuscularly by in vivo electroporation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Design of the p24CE DNA vaccine. (A) Alignment of the HXB2p24^(gag) protein sequences with the consensus clades A, B and C and theGroup M consensus, the Group M Center-of-Tree (COT-M) and the 7 CEincluded in p24CE1 and p24CE2. The ‘toggle’ amino acid differencesbetween the CE1 and CE2 sequences are indicated. (B) Localization of CEwithin the hexameric p24^(gag) structure. The p24^(gag) structure ismodified from Pornillos et al. [97] and shows the location of CE1-CE7(red), the toggle AA (blue) and the AA not included in the CE (black).The crystal structure of the hexamer was obtained from the www httpaddress ebi.ac.uk/pdbsum (C) Kyte-Dolittle hydrophobicity plots for twodifferent collinear arrangements of the (D) The p24CE (p24CE1 andp24CE2) proteins are composed of 7 CE arranged collinearly and linkedvia amino acid linkers. The secreted SP-p24CE contains the GM-CSF signalpeptide. MCP3-p24CE is a fusion protein with the Monocytechemoattractant protein 3 (MCP-3) chemokine. LAMP-p24CE is a fusion withthe lysosomal associated membrane protein 1 (LAMP-1),

FIG. 2: Expression of the p24CE plasmids upon transient transfection incultured cells. Plasmid DNA (1 μg) expressing different variants ofeither p24CE1 (left panel) or p24CE2 (right panel) proteins weretransfected in HEK293 cells. The cultures were harvested 24 hrs laterand proteins from equal amounts ( 1/250) from the cell-associated (toppanel) and extra-cellular (bottom panel) fractions were resolved on a12% NuPAGE Bis-Tris gel and analyzed by Western immunoblot using a goatanti-p24^(gag) antiserum and visualized using enhanced ECL. The membranecontaining the cell-associated fractions was also probed with anti-humanpan actin antibody to control for equal loading of the samples.

FIG. 3: Cellular responses in p24CE DNA vaccinated C57BL/6 mice. Micewere vaccinated using in vivo EP with 20 μg of the indicated p24CE1(left panel) Of p24CE2 (right panel) DNA plasmids (A, B) or withSP-p24CE1 DNA (C). Splenocytes from individual animals were stimulated(A) with the Group M Consensus peptide pool (15-mer peptides overlappingby 11 AA), (B) with the COT-M peptide pool (10-mea overlapping by 9 AA)consisting of the matching peptides of p24CE1 and p24CE2 proteins, and(C) with peptide pools representing the clade A, B, and C p55^(gag)sequences (15-mer peptides overlapping by 11 AA), as described inMaterials and Methods. The frequency of CE-specific IFN-γ producing CD4⁺(open bars) and CD8⁺ (filled bars) T cells was determined bypolychromatic flow cytometry. The mean and SEM are shown. Threeexperiments were performed and data from a representative experiment areshown.

FIG. 4: Mapping of the p24CE-induced cellular immune responses. Pooledsplenocytes from C57BL/6 mice (N=5) vaccinated with the indicated p24CE1(left panels) or p24CE2 (right panels) DNAs were stimulated with theGroup M Consensus peptide pools (15-mers overlapping by 11 AA) spanningthe individual CEs. The frequency of CE-specific IFN-γ producing T cellswas measured. CD4⁺ (open bars) and CD8⁺ (filled bars) Gag-specific Tcells are shown,

FIG. 5: Phenotypic and functional analysis of T cell responses generatedby p55^(gag) and p24CE DNA vaccination. (A) Mice (N=5/group) werevaccinated 3 times (week 0, 3 and 6) with 20 μg of a plasmid expressingHXB2 p55^(gag) (clade B) or 20 μg of a mixture of plasmids expressingSP-p24CE1 and SP-p24CE2. The mice were sacrificed 2 weeks after the lastimmunization. Three independent experiments were performed and arepresentative experiment is shown. (B) Pooled splenocytes werestimulated with Clade A, B or C peptide pools (15-mers) spanning thep24^(gag) region (left panel) and the Group M consensus peptide pool(right panel). The frequency of the CD4⁺ (open bars) and CD8⁺ (filledbars) p24^(gag)-specific IFN-γ producing T cells was determined (C) Thesplenocytes from the SP-24CE (left panel) and p55^(gag) (right panel)DNA vaccinated mice were stimulated with peptide pools specific for theindividual CEs. The frequency of the CD4⁺ (open bars) and CD8⁺ (filledbars) CE-specific IFN-γ producing T cells was determined. (D) Plotoverlays show the phenotypic and functional characterization of theantigen-specific T cells induced by SP-p24CE (left panels) and p55^(gag)(right panels) DNA vaccines upon stimulation with p24^(gag)-specificpeptide pool. Total T cells recovered from the spleen are shown as greycontours, and the antigen-specific IFN-γ⁺ T cells are overlaid as red(CD4⁺ T cells) or black (CD8⁺ T cells) dots. The plots show the CD4/CD8distribution (top panel), memory phenotype as determined by CD44/CD62Lstaining (middle panel) and TNFα/CD107a expression (bottom panel) amongthe T cells from vaccinated mice. The frequency of CD4 (red) and CD8(black) IFN-γ T lymphocytes is shown.

FIG. 6: Humoral immune responses in p24CE DNA vaccinated mice. (A)Anti-HIV-1 p24^(gag) antibodies were measured in plasma from p24CE andp55^(gag) DNA vaccinated C57BL/6 mice by a standard chide B p24^(gag)ELISA. The graphs show absorbance (optical density, OD) and pooledplasma samples dilutions from mice vaccinated with the different p24CE1plasmids (top panel), p24CE2 plasmids (middle panel), or p55^(gag) DNA(bottom panel). (B) Humoral responses induced upon SP-p24CE or p55^(gag)DNA vaccination in mice were analyzed by Western immunoblot assays. Themembranes contain p24^(gag) protein collected from supernatants ofHEK293 cells transfected with 5 μg of the infectious molecular clonepNL4-3 (lane 1) or the p24CE proteins collected from the cell-associatedfractions of cells transfected with SP-p24CE1 and SP-p24CE2 plasmids(lanes 2 and 3, respectively). The membranes were probed with plasma(1:5000 dilution) from mice vaccinated with a mixture of SP-p24CE1&2DNAs (top panel) or p55^(gag) DNA (bottom panel) followed by anti-mouseIgG-HRP labeled antibody and visualized by ECL. (C) Detection of humoralresponses to full-length p55^(gag) in mice vaccinated with p24CE orp55^(gag) DNA by Western immunoblot assay. The p55^(gag) proteins wereobtained from HEK293 cells transfected with 0.5 μg of RNA/codonoptimized plasmids expressing unprocessed p55^(gag) from clades A, B andC or COT-M, respectively. The proteins were resolved on 10% NuPAGEBis-Tris gels, and the membranes were probed with plasma (dilution1:200) from mice immunized with DNAs expressing the secreted p24CEproteins SP-p24CE1 (top panel), SP-p24CE2 (middle panel) and p55^(gag)(bottom panel).

FIG. 7: p55^(gag) DNA vaccination of macaques induces poor CE-specificcellular immune responses. (A) Alignment of the amino acid (AA) sequenceof the 7 CE represented in the p24CE1 and p27CE2 proteins with HXB2p24^(gag) protein. The toggled AA in each CE is shown. The numbering ofthe AA in HXB2 p24^(gag) protein is according to the HIV data base atthe www site hiv.lanl.gov/. (B) Both p24^(gag). . . specific andCE-specific T cell responses were measured at 2 weeks after the lastvaccination from 11 macaques, which were immunized with plasmid DNAencoding HIV Gag. (C) Mapping of the individual CE-specific responses in5 (of 11) macaques that had responses to CE (panel B). The frequency ofIFN-γ⁺ T cells specific for each of the 7 CE is shown. Open bars: CD4⁺ Tcells; filled bars: CD8⁺ T cells. (D). Dot plots showing the IFN-γ⁺ andgranzyme B (GzmB) production of the CE-specific CD4⁺ (red) and CD8⁺(black) T cells from the 5 macaques shown in panel C.

FIG. 8: CE-specific cellular immune responses upon vaccination ofmacaques with p24CE DNAs. (A) Macaques were vaccinated with p24CE DNAand the frequency of CE-specific T cells was measured 2 weeks after the2^(nd) vaccination (EP2wk2). IFN-γ⁺ CD4⁺ (open bars) and CD8⁺ (filledbars) T cells are shown. (B) Mapping of individual CE-specific responsesin the 6 immunized macaques from panel A. The frequency of IFN-γ⁺ CD4⁺(open bars) and CD8⁺ (filled bars) T cells specific for each CE isshown. (C) Phenotypic characterization of the CE-specific CD4⁺ (red) andCD8⁺ (black) T cells with CD28⁺CD95⁺ (central memory) or CD28⁻CD95⁺(effector memory phenotype) is shown in the top panel; IFN-γ⁺ andgranzyme B expression is shown in the bottom panel.

FIG. 9: induction of broad and polyfunctional T cell responses in p24CEvaccinated macaques. (A) The number of CE recognized per animal inmacaques vaccinated with p24CE DNA and p55^(gag) DNA are shown. (B)Frequency of CE-specific polyfunctional T cells was evaluated by theirability to produce IFN-γ, TNF-α, CD107a and granzyme B (GzmB). The datafrom one representative macaque from each group is shown: M437 (toppanel) vaccinated with p24CE DNA; P574 (middle panel) vaccinated withp55^(gag) DNA. The pie charts (right) show the proportion ofpolyfunctional responses in these macaques. Frequency of IFN-γ⁺, TNF-α⁺,CD107a⁺ and GzmB⁺ polyfunctional CE-specific T cells (4 functions) as %of total T cells in macaques vaccinated with p24CE DNA and p55^(gag)DNA, respectively, are shown (bottom panel). Median values areindicated.

FIG. 10: Boosting of p24CE DNA primed macaques with p55^(gag) DNAincreases CE-specific cellular responses. (A) Vaccination schedule ofgroup 1 with p24CE plasmid DNAs (EP1, EP2) followed by the heterologousp55^(gag) DNA boost (EP3) and group 2 with p55^(gag) DNA (EP1, EP2)followed by the heterologous p24CE DNA boost (EP3). (B) Frequency of theCE-specific IFN-γ⁺ T cells in both groups before (EP2wk2) and after theheterologous boost (EP3wk2). (C) Frequency of IFN-γ⁺ TNF-α⁺CD107a⁺GzmB⁺polyfunctional CE-specific T cells (4-function) in groups 1 and 2 beforeand after the heterologous boost.

FIG. 11: Mapping of CE-specific T cell responses before and afterheterologous boost. The CE-specific responses were mapped as describedfor FIG. 2B. The plots show comparisons of the responses upon p24CE DNAvaccination followed by p55^(gag) DNA boost (group 1, A) and uponp55^(gag) DNA vaccination followed by p24CE boost (group 2, B). Thepercentage of IFN-γ⁺ CD4⁺ (open bars) and CD8⁺ (filled bars) T cellsspecific for each CE is shown.

FIG. 12: Humoral immune responses upon p24CE DNA vaccination are boostedby p55^(gag) DNA vaccination. (A) Reciprocal p24^(gag) binding antibodyendpoint titers (log) measured in plasma by ELISA at the indicated limepoints for macaques in group 1 and group 2 before and after theheterologous boosts. (B) Western immunoblot analysis was used to testthe reactivity of the vaccine-induced antibodies from macaques in group1 and 2 to p24^(gag) and the p24CE proteins. The membranes containeither p24^(gag) protein (lanes 1 and 4), p24CE1 (lanes 2 and 5) orp24CE2 (lanes 3 and 6) and were probed with plasma from macaques fromgroup 1 (dilution 1:2000) and group 2 (dilution 1:500) collected before(EP2wk2; lanes 1-3) and after (EP3wk2, lanes 4-6) the heterologousboost.

FIG. 13: Sequences and configurations of p24CEc polypeptides and p24CEdpolypeptides.

FIG. 14: Plasmid map (A) and sequence (B) of plasmid 306H that encodesp24 CE1+p24 CE2.

FIG. 15: Plasmid map (A) and sequence (B) of plasmid 202H that encodesLAMP-p24CE2.

FIG. 16: Plasmid map (A) and sequence (B) of plasmid 191H that encodesLAMP-p24CE1.

FIG. 17: Plasmid map (A) and sequence (B) of plasmid 230H that encodesMCP3-p24CE1.

FIG. 18: Plasmid map (A) and sequence (B) of plasmid 231H that encodesMCP3-p24CE2.

FIG. 19: Plasmid map (A) and sequence (B) of plasmid 235H that encodesSP-p24CE2.

FIG. 20: Illustrative regimens for administering conserved element Gagvaccines and full-length p55gag vaccine.

FIG. 21: Illustrative data showing cellular immune responses before andafter the boost. Cellular immune responses were measured with peptides(15-mer overlapping by 11 amino acids) spanning the complete p24gag.

FIG. 22: Analysis of the responses to individual CE. The responses toeach CE were mapped in all the animals using CE-specific peptidesmixture of 10-mer peptide overlapping by 9 amino acids and 15-meroverlapping by 11 amino acids) for each CE. The number of CE that showedpositive responses per animal are shown,

FIG. 23: Different vaccine strategies induced similar levels of p27gagantibody responses. Binding antibody titers were measured in the plasmaby ELISA.

DETAILED DESCRIPTION OF THE INVENTION Definitions

A “conserved element” as used herein refers to a protein sequence thatis conserved across a protein that has high sequence diversity innature, e.g., a viral protein such as an gag. The conserved element neednot have 100% sequence identity across the diversity of naturallyoccurring sequence of the protein, but the sequence variability in thenaturally occurring sequences is low, e.g., less than 20%. In someembodiments, the sequence variability is less than 10%. A conservedelement is usually eight amino acids, or greater, e.g., 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. Typically aconserved element is less than 50 amino acids in length and often isless than 40 or less than 30 amino acids. In some embodiments, aconserved element is less than 25 amino acids in length.

A “nucleic acid vaccine” as used herein includes both naked DNAvaccines, e.g., plasmid vaccine, and viral vector-based nucleic acidsvaccines that are comprised by a viral vector and/or delivered as viralparticles.

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Degenerate codon substitutions can beachieved by generating sequences in which the third position of one ormore selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Balzer et al., Nucleic Acid Res. 19:5081 (1991);Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al.,Mol. Cell. Probes 8:91-98 (1994)). The term “nucleic acid” is usedinterchangeably with gene, cDNA, oligonucleotide, and polynucleotide. A“nucleic acid” encompasses RNA as well as DNA.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(e.g., about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or higher identity over a specified region is polypeptide sequencecomprising conserved elements), when compared and aligned for maximumcorrespondence over a comparison window or designated region) asmeasured using a BLAST or BLAST 2.0 sequence comparison algorithms withdefault parameters described below, or by manual alignment and visualinspection (see, e.g., NCBI web site or the like). Such sequences arethen said to be “substantially identical.” This definition also refersto, or can be applied to, the compliment of a test sequence. Thedefinition also includes sequences that have deletions and/or additions,as well as those that have substitutions. As described below, thepreferred algorithms can account for gaps and the like. Preferably,identity exists over a region that is at least about 25, 50, 75, 100,150, 200 amino acids or nucleotides in length, and oftentimes over aregion that is 225, 250, 300, 350, 400, 450, 500 amino acids ornucleotides in length or over the full-length of an amino acid ornucleic acid sequences.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A preferred example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLASTalgorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. BLAST software is publicly available through theNational Center for Biotechnology Information on the worldwide web atncbi.nlm.nih.gov/. Both default parameters or other non-defaultparameters can be used. The BLASTN program (for nucleotide sequences)uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5,N=−4 and a comparison of both strands. For amino acid sequences, theBLASTP program uses as defaults a wordlength of 3, and expectation (E)of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc.Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation(E) of 10, M=5, N=−4, and a comparison of both strands.

The term “operably linked” refers to a functional linkage between afirst nucleic acid sequence and a second nucleic acid sequence, suchthat the first and second nucleic acid sequences are transcribed into asingle nucleic acid sequence. Operably linked nucleic acid sequencesneed not be physically adjacent to each other. The term “operablylinked” also refers to a functional linkage between a nucleic acidexpression control sequence (such as a promoter, or array oftranscription factor binding sites) and a transcribable nucleic acidsequence, wherein the expression control sequence directs transcriptionof the nucleic acid corresponding to the transcribable sequence.

Amino acids can be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,can be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” as used herein applies to amino acidsequences. One of skill will recognize that individual substitutions,deletions or additions to a nucleic acid, peptide, polypeptide, orprotein sequence which alters, adds or deletes a single amino acid or asmall percentage of amino acids in the encoded sequence is a“conservatively modified variant” where the alteration results in thesubstitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention. The following eight groups eachcontain amino acids that are conservative substitutions for one another:

-   -   1) Alanine (A), Glycine (G);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);    -   7) Serine (S), Threonine (T); and    -   8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins        (1984)).

The terms “mammal” or “mammalian” refer to any animal within thetaxonomic classification mammalia. A mammal can refer to a human or anon-human primate. A mammal can refer to a domestic animal, includingfor example, canine, feline, rodentia, including lagomorpha, murine,rattus, Cricetinae (hamsters), etc. A mammal can refer to anagricultural animal, including for example, bovine, ovine, porcine,equine, etc.

The terms “enhanced immune response” or “increased immune response” asused herein refers to an immune response to the conserved elementnucleic acid and full-length, or substantially full length protein thatare administered, where the immune response is increased in comparisonto when only the conserved element vaccine or full-length protein isadministered. An “enhanced immune response” may include increases in thelevel of immune cell activation and/or an increase in the duration ofthe response and/or immunological memory as well as an improvement inthe kinetics of the immune response. The increase can be demonstrated byeither a numerical increase, e.g., an increased in levels of antibody ina particular time frame, as assessed in an assay to measure the responseassay or by prolonged longevity of the response.

The terms “treating” and “treatment” refer to delaying the onset of,retarding or reversing the progress of, or alleviating or preventingeither the disease or condition to which the term applies, or one ormore symptoms of such disease or condition.

An “antigen” refers to a molecule, typically a protein molecule in thecurrent invention, containing one or more epitopes (either linear,conformational or both) that will stimulate a host's immune system tomake a Immoral and/or cellular antigen-specific response. The term isused interchangeably with the term “immunogen.” Normally, an epitopewill comprise between about 7 and 15 amino acids, such as, 9, 10, 12 or15 amino acids. The term “antigen” includes both subunit antigens,(i.e., antigens which are separate and discrete from a whole organismwith which the antigen is associated in nature), as well as inactivatedorganisms, such as viruses.

Introduction

The invention is based in part, on the discovery that administration ofone or more nucleic acids encoding a polypeptide comprising conservedelements from a protein to a subject in conjunction with administrationof a nucleic acid encoding the full-length protein, or substantiallyfull-length protein, enhances the immune response to the conservedelement sequences. The protein can be any protein, but is typically aviral protein that exhibits sequence diversity in naturally occurringvariants. In some embodiments, the viral protein is a retrovirusprotein, such as a lentiviral protein. In some embodiments, the viralprotein is a retroviral gag or env protein.

In some embodiments, administration of the nucleic acid encoding thefull-length protein, or substantially full-length protein, followsadministration of a conserved element nucleic acid construct. Thus, theinvention further provides methods of inducing an immune responsecomprising sequential administration of at least one conserved elementnucleic acid construct followed by administration of a nucleic acidconstruct comprising substantially a full length protein from which theconserved elements are derived.

Conserved Element Nucleic Acid Constructs

Conserved elements of a protein sequence can be determined using knownmethods. For examples U.S. Patent Application Publication No.20110269937, which is incorporated by reference, describes methods ofevaluating protein sequences that exhibit natural variability toidentify regions that are conserved using computational methods.

A conserved element nucleic acid construct is typically generated bylinking nucleic acid sequences that encode multiple conserved elementsthat target conserved sequence that are present within all or a highpercentage, e.g., at least 80%, at least 90%, or at least 95%, orgreater, of the naturally occurring variants of the protein in apopulation. In typical embodiments, a conserved element is from a regionof a protein that when mutated, has deleterious effects on the functionof the protein. In typical embodiments, a conserved element does notcomprise an amino acid sequence that does not occur in a naturallyoccurring variant, i.e., the conserved element does not contain aminoacid substitutions that would result in a sequence that has not beenidentified in a naturally occurring variant.

In some embodiments a immunogenic compositions employed in the inventionrelates to a viral protein, e.g., a retrovirus protein such as Gag.Conserved elements of Gag have been identified (see, e.g., U.S. PatentApplication Publication No. 20110269937; Rolland et al., PLoS Pathog 3:e157, 2007; Motile et al., PLoS One 7: e29717, 2012).

In some embodiments, the nucleic acid construct encoding the conservedelement polypeptide encodes a polypeptide that comprises at least one,two, three, four, five, six, or seven conserved elements set forth inFIG. 1. In some embodiments, the nucleic acid construct encodes apolypeptide that comprises at least 8, typically, at least 9, 10, 11,12, 14, 15, 16, 17, 18, 19, 20, or more consecutive amino acids from theconserved elements set forth in FIG. 1; or in SEQ ID NOS:1-14, 32, 33.40 and 41.

In typical embodiments, more than one nucleic acid construct encodingthe conserved elements is used where one construct encodes a first setof conserved elements and the second construct encodes a second set ofconserved elements where one or more elements, often each of theconserved elements, of the second set of conserved elements differs fromthe first set by 3 or fewer amino acids. The residues where thesequences differ, however, are at sites of naturally occurringvariation, so that each of the conserved elements in the first andsecond sets corresponds to a naturally occurring protein sequence. Insome embodiments, each element of the second set is at least 80% or atleast 90% identical to the corresponding element in the first set ofconserved sequences. The nucleic acid construct encoding the first setof conserved elements and the nucleic acid construct encoding the secondset of conserved elements may be present in the same vector or differentvectors.

Each conserved element useful for an immunogenic nucleic acidadministered in accordance with the methods of the invention istypically less than 30 amino acids in length. In some embodiments, theconserved element is less than 25, 24, 23, 22, 21, 20, or 15 amino acidsin length. In some embodiments, the conserved element is 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids inlength.

In the present invention, the conserved elements contained within theconserved elements are not contiguous in the native protein sequence.The individual conserved elements are typically joined to one another inthe nucleic acid construct by a peptide such as an alanine linker.Linker sequences are well known in the art. Typical peptide linkersequences contain Gly, Ser, Ala and Thr residues. Useful linkers includeglycine-serine polymers; glycine-alanine polymers; alanine-serinepolymers. In some embodiments, the linker is AA, AAAE, AAAA, AAK, AG,AA, LAK, AAK, AAAAL, and the like.

The conserved elements may be present in any order in the construct,they need not occur in the order of the naturally occurring sequence.For example, a conserved element that occurs toward the N-terminus of aprotein may be encoded at region of the construct encoding theC-terminal end.

In some embodiments, a nucleic acid encoding a conserved elementpolypeptide for use in the invention encodes a polypeptide thatcomprises the conserved elements set forth in SEQ ID NOS:1-7. In someembodiments, such a nucleic acid construct encodes a polypeptidecomprising the amino acid sequence of SEQ ID NO:15 (“Core1”). In someembodiments, a nucleic acid encoding a conserved element encodes apolypeptide comprising the conserved elements set forth in SEQ IDNOS:8-14. In some embodiments, such a nucleic acid construct encodes apolypeptide that comprises the amino acid sequence of SEQ ID NO:16(“Core2”). In some embodiments, a nucleic acid construct encoding apolypeptide comprising SEQ ID NO:15 is administered with a nucleic acidconstruct encoding a polypeptide comprising SEQ ID NO:16.

In some embodiments, a nucleic acid encoding a conserved element encodesa conserved elements set forth in SEQ ID NOS: 3, 4, 5, 6, 32, 33, 40 or41, or a variant thereof that differs by 1 amino acid. In someembodiments, a variant of SEQ ID NO:33 may differ at 1, 2, or 3 aminoacids. In some embodiments, the conserved element polypeptide comprisesa sequence set forth in FIG. 13.

In the present invention, a conserved element nucleic acid construct isadministered in conjunction with the full-length protein, orsubstantially full-length protein, from which the conserved elements areobtained. In the context of the present invention, “substantiallyfull-length” refers to the region of the protein that includes all ofthe conserved elements, i.e., a sufficient length of the naturallyoccurring protein is provided that includes of the conserved elementsthat are used in the conserved element construct.

The nucleic acid encoding the full-length protein may be administeredconcurrently with the conserved element vaccine. In some embodiments, afull-length protein may be administered as the priming vaccine prior toadministration of one or more conserved element constructs, which areadministered as a boost. In preferred embodiments, one or more nucleicacids encoding conserved elements are administered as the prime and thenucleic acid encoding the full-length protein is administered as aboost. The boost is typically administered anywhere from two weeks toone, two, three, or four months, or longer, following, administration ofthe initial vaccine.

Often, the nucleic acid constructs encoding the conserved elementsand/or full-length protein are one or more purified nucleic acidmolecules, for example, one or more plasmid-based vectors (“naked” DNA).

In some embodiments, the nucleic acid component may comprise vectorsthat encode the antigen of interest where the vector is contained withina virus. Viral delivery systems include adenovirus vectors,adeno-associated viral (AAV) vectors, herpes viral vectors, retroviralvectors, poxviral vectors, or lentiviral vectors. Methods ofconstructing and using such vectors are well known in the art.

Recombinant viruses in the pox family of viruses can be used fordelivering the nucleic acid molecules encoding the antigens of interest.These include vaccinia viruses and avian poxviruses, such as the fowlpoxand canarypox viruses. Methods for producing recombinant pox viruses areknown in the art and employ genetic recombination. See, e.g., WO91/12882; WO 89/03429; and WO 92/03545. A detailed review of thistechnology is found in U.S. Pat. No. 5,863,542. Representative examplesof recombinant pox viruses include ALVAC, TRW/AC, and NYVAC.

A number of adenovirus vectors have also been described that can be usedto deliver one or more of the nucleic acid components of the vaccine.(Haj-Ahmad and Graham, J. Virol. (1986) 57:267-274; Bett et al., J.Virol. (1993) 67:5911-5921; Mittereder et al., Human Gene Therapy (1994)5:717-729; Seth et al., J. Virol. (1994) 68:933-940; Barr et al., GeneTherapy (1994) 1:51-58; Berkner, K. L. BioTechniques (1988) 6:616-629;and Rich et al., Human Gene Therapy (1993) 4:461-476). Additionally,various adeno-associated virus (AAV) vector systems have been developedfor gene delivery. AAV vectors can be readily constructed usingtechniques well known in the art. See, e.g., U.S. Pat. Nos. 5,173,414and 5,139,941; International Publication Nos. WO 92/01070 (published 23Jan. 1992) and WO 93/03769 (published 4 Mar. 1993); Lebkowski et al.,Molec. Cell. Biol. (1988) 8:3988-3996; Vincent et al., Vaccines 90(1990) (Cold Spring Harbor Laboratory Press); Carter, B. J. CurrentOpinion in Biotechnology (1992) 3:533-539; Muzyczka, N. Current Topicsin Microbiol. and Immunol. (1992) 158:97-129; Kotin, R. M. Human GeneTherapy (1994) 5:793-801; Shelling and Smith, Gene Therapy (1994)1:165-169; and Zhou et al., J. Exp. Med. (1994) 179:1867-1875.

Retroviruses also provide a platform for gene delivery systems. A numberof retroviral systems have been described (U.S. Pat. No. 5,219,740:Miller and Rosman, BioTechniques (1989) 7:980-990; Miller, A. D., HumanGene Therapy (1990) 1:5-14; Scarpa et al., Virology (1991) 180:849-852;Burns et al., Proc. Natl. Acad. Sci, USA (1993) 90:8033-8037; andBoris-Lawrie and Temin, Cur. Opin. Genet. Develop. (1993) 3:102-109.

Molecular conjugate vectors, such as the adenovirus chimeric vectorsdescribed in Michael et al., J. Biol. Chem. (1993) 268:6866-6869 andWagner et al., Proc. Natl. Acad. Sci, USA (1992) 89:6099-6103, can alsobe used for gene delivery.

Members of the Alphavirus genus, such as, but not limited to, vectorsderived from the Sindbis, Semliki Forest, and Venezuelan EquineEncephalitis viruses, can also be used as viral vectors to deliver oneor more nucleic acid components of the nucleic acid/protein combinationvaccines of the invention. For a description of Sindbis-virus derivedvectors useful for the practice of the instant methods, see, Dubensky etal., J. Virol, (1996) 70:508-519; and International Publication Nos. WO95/07995 and WO 96/17072; as well as, Dubensky, Jr., T. W., et al., U.S.Pat. No. 5,843,723, issued Dec. 1, 1998, and Dubensky, Jr., T. W., U.S.Pat. No. 5,789,245, issued Aug. 4, 1998).

Expression Constructs Encoding Fusion Polypeptides Comprising aDegradation Signal or Signal Peptide Sequence

In some embodiments, a nucleic acids encoding a conserved elementvaccine encodes a form in which the conserved element is fused to asequence to enhance the immune response, such as a signal peptidesequence or a sequence that targets the protein for lysosomaldegradation. Such embodiments typically results in enhanced immuneresponses in comparison to embodiments where the conserved elementvaccine is not fused to a signal peptide or degradation signal.

Lysosomal Targeting Sequence

In other embodiments, signals that target proteins to the lysosome mayalso be employed. For example, the lysosome associated membraneproteins1 and 2 (LAMP-1 and LAMP-2) include a region that targetsproteins to the lysosome. Examples of lysosome targeting sequences areprovided, e.g., in U.S. Pat. Nos. 5,633,234; 6,248,565; and 6,294,378.

Destabilizing sequences present in particular proteins are well known inthe art. Exemplary destabilization sequences include c-myc aa 2-120;cyclin A aa 13-91; Cyclin B aa 13-91; IkBα aa 20-45: β-Catenin aa 19-44;β-Catenin aa 18-47, c-Jun aa1-67; and c-Mos aa1-35; and fragments andvariants, of those segments that mediate destabilization. Such fragmentscan be identified using methodology well known in the art. For example,polypeptide half-life can be determined by a pulse-chase assay thatdetects the amount of polypeptide that is present over a time courseusing an antibody to the polypeptide, or to a tag linked to thepolypeptide. Exemplary assays are described, e.g., in WO02/36806, whichis incorporated by reference.

Variants of such sequences, e.g., that have at least 90% identity,usually at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, orgreater, identity to the sequences noted above, e.g., a LAMP degradationsequence, can be employed in this invention, e.g., for fusion to an gagconserved element polypeptide.

Additional degradation signals that can be used to modify retroviralantigens, e.g. HIV antigens in accordance with the invention include theF-box degradation signal, such as the F-BOX signal 47aa (182-228) fromprotein beta-TrCP (Liu, et al., Biochem Biophys Res Comm. 313:1023-1029,2004). Accordingly, in some embodiments, an expression vector for use inthe invention may encode a fusion protein where an F-box degradationsignal is attached to an HIV antigen, e.g., gag.

Targeting to the Proteasome and Other Degradation Signals

Many polypeptide sequences that target a protein for degradation areknown in the art. One example of destabilizing sequences are so-calledPEST sequences, which are abundant in the amino acids Pro, Asp, Glu,Ser, Thr (they need not be in a particular order), and can occur ininternal positions in a protein sequence. A number of proteins reportedto have PEST sequence elements are rapidly targeted to the 26Sproteasome. A PEST sequence typically correlates with a) predictedsurface exposed loops or turns and b) serine phosphorylation sites, e.g.the motif S/TP is the target site for cyclin dependent kinases.

Additional destabilization sequences relate to sequences present in theN-terminal region. In particular the rate of ubiquitination, whichtargets proteins for degradation by the 26S proteasome can be influencedby the identity of the N-terminal residue of the protein. Thus,destabilization sequences can also comprise such N-terminal residues,“N-end rule” targeting (see, e.g., Tobery et al., J. Exp. Med.185:909-920).

Other targeting signals include the destruction box sequence that ispresent, e.g., in cyclins. Such a destruction box has a motif of 9 aminoacids, R1(A/T)2(A)3L4(G)5X6(I/V)7(G/T)8(N)9, in which the onlyinvariable residues are R and L in positions 1 and 4, respectively. Theresidues shown in brackets occur in most destruction sequences. (see,e.g., Hershko & Ciechanover, Annu. Rev. Biochem. 67:425-79, 1998). Inother instances, destabilization sequences lead to phosphorylation of aprotein at a serine residue (e.g., Iκbα).

In some embodiments, a conserved element polypeptide of the invention isfused to a LAMP degradation sequence. For example, the methods of theinvention may employ a polypeptide in which SEQ ID NO:15 or SEQ ID NO:16is fused to a LAMP degradation sequence.

Expression Constructs that Encode Secreted Fusion Proteins

A secretory polypeptide in the context of this invention is apolypeptide signal sequence that results in secretion of the protein towhich it is attached. In some embodiments, the secretory polypeptide isa chemokine, cytokine, or lymphokine, or a fragment of the chemokine,cytokine, or lymphokine that retains immunostimulatory activity.Examples of chemokines secretory polypeptides include MCP-3 and IP-10.In other embodiments, the secretory polypeptide is a polypeptide signalsequence from a secreted protein such as tissue plasminogen activator(tPA) protein, growth hormone, GM-CSF, a cytokine, or an immunoglobulinprotein. Constructs encoding secretory fusion proteins are disclosed,e.g., in WO02/36806.

In some embodiments, the signal peptide is a GM-CSF sequence, e.g., amammalian GM-CSF sequence such as a human GM-CSF signal peptidesequence.

In some embodiments, a secretory signal for use in the invention isMCP-3 amino acids 33-109, e.g., linked to IP-10 secretory peptide.

In some embodiments, a conserved element polypeptide is joined to aGM-CSF signal peptide sequence. For example, in some embodiments, themethods of the invention may employ a polypeptide comprising SEQ IDNO:15 fused to a GM-CSF signal peptide and/or a polypeptide comprisingSEQ ID NO:16 fused to a GM-CSF signal peptide.

Similarly, an expression construct encoding a full-length polypeptide,e.g., a construct that encodes p55 gag, may also be modified with adegradation sequence and/or a secretory sequence. Moreover, more thanone construct encoding the full-length polypeptide, e.g, p55gag may beadministered. For example a construct in which p55 gag is fused to asignal polypeptide such as GM-CSF may be used in conjunction with aconstruct in which p55gag is fused to a degradation sequence, such asLAMP.

Additional Properties of Expression Constructs

Within each expression cassette, sequences encoding an antigen for usein the nucleic acid vaccines of the invention will be operably linked toexpression regulating sequences. “Operably linked” sequences includeboth expression control sequences that are contiguous with the nucleicacid of interest and expression control sequences that act in trans orat a distance to control the gene of interest. Expression controlsequences include appropriate transcription initiation, termination,promoter and enhancer sequences; efficient RNA processing signals suchas splicing and polyadenylation signals; sequences that stabilizecytoplasmic mRNA; sequences that promote RNA export (e.g., aconstitutive transport element (CTE), a RNA transport element (RTE), orcombinations thereof; sequences that enhance translation efficiency(e.g., Kozak consensus sequence); sequences that enhance proteinstability; and when desired, sequences that enhance protein secretion.

Any of the conventional vectors used for expression in eukaryotic cellsmay be used for directly introducing nucleic acids into tissue.Expression vectors containing regulatory elements from eukaryoticviruses are often used in eukaryotic expression vectors. Such regulatoryelements include, e.g., human CMV, simian CMV, viral LTRs, and the like.Typical vectors may comprise, e.g. those with a human CMV promoter,bovine growth hormone polyA site and an antibiotic resistance gene forselective growth in bacteria.

Other expression vector components are well known in the art, including,but not limited to, the following: transcription enhancer elements,transcription termination signals, polyadenylation sequences, splicesites, sequences for optimization of initiation of translation, andtranslation termination sequences.

In some embodiments, the nucleic acid component may comprises one ormore RNA molecules, such as viral RNA molecules or mRNA molecules thatencode the antigen of interest.

In typical embodiments, the nucleic acid constructs are codon-optimizedfor expression.

In the present invention, a “nucleic acid” molecule can include cDNA,and genomic DNA sequences, RNA, and synthetic nucleic acid sequences.Thus, “nucleic acid” also encompasses embodiments in which analogs ofDNA and RNA are employed.

An immunogenic composition of the invention can be administered as oneor more constructs. For example, where two sets of conserved elementsare employed, e.g., conserved element polypeptides Core1 and Core2, orconserved element polypeptides p2CE1c and p24CE2c or conserved elementpolypeptides p2CE1d and p24CE2d, a nucleic acid construct can encodeboth sets, or each set may be encoded by a separate expression vector.Thus, the expression constructs administered in accordance with theinvention may be administered as multiple expression vectors, or as oneor more expression vectors encoding multiple expression units, e.g., adiscistronic, or otherwise multicistronic, expression vectors. Forexample, an expression vector may be employed that encodes both SEQ IDNO:15 and SEQ ID NO:16 or multiple expression vectors may be employedwhere SEQ ID NO:15 is encoded by one vector and SEQ ID NO:16 is encodedby another vector.

Preparation of Immunogenic Compositions

In the methods of the invention, the nucleic acid component is oftendirectly introduced into the cells of the individual receiving theimmunogenic composition. This approach is described, for instance, inWolff et. al., Science 247:1465 (1990) as well as U.S. Pat. Nos.5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; and WO98/04720. Examples of DNA-based delivery technologies include, “nakedDNA”, facilitated (bupivicaine, polymers, peptide-mediated) delivery,and cationic lipid complexes or liposomes. The nucleic acids can beadministered using ballistic delivery as described, for instance, inU.S. Pat. No. 5,204,253 or pressure (see, e.g., U.S. Pat. No.5,922,687). Using this technique, particles comprised solely of DNA areadministered, or in an alternative embodiment, the DNA can be adhered toparticles, such as gold particles, for administration.

In some embodiments, e.g, where a nucleic acid component of theinvention is encoded by a viral vector, the nucleic acid component canbe delivered by infecting the cells with the virus containing thevector. This can be performed using any delivery technology, e.g., asdescribed in the previous paragraph.

In some embodiments, the immunogenic compositions of the invention areadministered by injection or electroporation, or a combination ofinjection and electroporation,

Assessment of Immunogenic Response

To assess a patient's immune system during and after treatment and tofurther evaluate the treatment regimen, various parameters can bemeasured. Measurements to evaluate vaccine response include: antibodymeasurements in the plasma, serum, or other body fluids; and analysis ofin vitro cell proliferation in response to a specific antigen,indicating the function of CD4+ cells. Such assays are well known in theart. For example, for measuring CD4+ T cells, many laboratories measureabsolute CD4+ T-cell levels in whole blood by a multi-platform,three-stage process. The CD4+ T-cell number is the product of threelaboratory techniques: the white blood cell (WBC) count; the percentageof WBCs that are lymphocytes (differential); and the percentage oflymphocytes that are CD4+ T-cells. The last stage in the process ofmeasuring the percentage of CD4+ T-lymphocytes in the whole-blood sampleis referred to as “immunophenotyping by flow cytometry. Systems formeasuring CD4+ cells are commercially available. For example BectonDickenson's FACSCount System automatically measure absolutes CD4+, CD8+,and CD3+ T lymphocytes.

Other measurements of immune response include assessing CD8+ responses.These techniques are well known. CD8+ T-cell responses can be measured,for example, by using tenamer staining of fresh or cultured PBMC (see,e.g., Altman, et al., Proc. Natl. Acad. Sci. USA 90:10330, 1993; Altman,et al., Science 274:94, 1996), or γ-interferon release assays such asELISPOT assays (see, e.g., Lalvani, et al., J. Exp. Med. 186:859, 1997;Dunbar, et al., Curr. Biol. 8:413, 1998; Murali-Krishna, et al.,Immunity 8:177, 1998), or by using functional cytotoxicity assays.

Viral Titer

Viremia is measured by assessing viral titer in a patient. There are avariety of methods of perform this. For example, plasma HIV RNAconcentrations can be quantified by either target amplification methods(e.g., quantitative RT polymerase chain reaction [RT-PCR], Amplicor HIVMonitor assay, Roche Molecular Systems; or nucleic acid sequence-basedamplification, [NASBA®], NucliSensTM HIV-1 OT assay, Organon Teknika) orsignal amplification methods (e.g., branched DNA [bDNA], QuantiplexTMHIV RNA bDNA assay, Chiron Diagnostics). The bDNA signal amplificationmethod amplifies the signal obtained from a captured HIV RNA target byusing sequential oligonucleotide hybridization steps, whereas the RT-PCRand NASBA® assays use enzymatic methods to amplify the target HIV RNAinto measurable amounts of nucleic acid product. Target HIV RNAsequences are quantitated by comparison with internal or externalreference standards, depending upon the assay used.

Administration of DNA Constructs

The DNA vectors are formulated for pharmaceutical administration. Whileany suitable carrier known to those of ordinary skill in the art may beemployed in the pharmaceutical compositions of this invention, the typeof carrier will vary depending on the mode of administration. Forparenteral administration, including intranasal, intradermal,subcutaneous or intramuscular injection or electroporation, the carrierpreferably comprises water, saline, and optionally an alcohol, a fat, apolymer, a wax, one or more stabilizing amino acids or a buffer. Generalformulation technologies are known to those of skill in the art (see,for example, Remington: The Science and Practice of Pharmacy (20thedition), Gennaro, ed., 2000, Lippincott Williams & Wilkins; InjectableDispersed Systems: Formulation, Processing And Performance, Burgess,ed., 2005, CRC Press; and Pharmaceutical Formulation Development ofPeptides and Proteins, Frkjr et al., eds., 2000, Taylor & Francis).

Naked DNA can be administered in solution (e.g., a phosphate-bufferedsaline solution) by injection, usually by an intra-arterial,intravenous, subcutaneous or intramuscular route. In general, the doseof a naked nucleic acid composition is from about 10 μg, to 10 mg for atypical 70 kilogram patient. Subcutaneous or intramuscular doses fornaked nucleic acid (typically DNA encoding a fusion protein) will rangefrom 0.1 mg to 50 mg for a 70 kg patient in generally good health.

DNA immunogenic compositions can be administered once or multiple limes.DNA vaccination is performed More than once, for example, 2, 3, 4, 5, 6,7, 8, 10, 15, 20 or more times as needed to induce the desired response(e.g., specific antigenic response or proliferation of immune cells).Multiple administrations can be administered, for example, bi-weekly,weekly, bi-monthly, monthly, or more or less often, as needed, for atime period sufficient to achieve the desired response.

The nucleic acid constructs in accordance with the invention areadministered to a mammalian host. The mammalian host usually is a humanor a primate. In some embodiments, the mammalian host can be a domesticanimal, for example, canine, feline, lagomorpha, rodentia, rattus,hamster, murine. In other embodiment, the mammalian host is anagricultural animal, for example, bovine, ovine, porcine, equine, etc.

Immunogenic compositions containing the DNA expression constructs can beformulated in accordance with standard techniques well known to thoseskilled in the pharmaceutical art. Such compositions can be administeredin dosages and by techniques well known to those skilled in the medicalarts taking into consideration such factors as the age, sex, weight, andcondition of the particular patient, and the route of administration.

Iii therapeutic applications, the vaccines are administered to a patientin an amount sufficient to elicit a therapeutic effect, e.g., a CD8⁺,CD4⁺, and/or antibody response to the HIV-1 antigens encoded by thevaccines that at least partially arrests or slows symptoms and/orcomplications of HIV infection. An amount adequate to accomplish this isdefined as “therapeutically effective dose.” Amounts effective for thisuse will depend on, e.g., the particular composition of the vaccineregimen administered, the manner of administration, the stage andseverity of the disease, the general state of health of the patient, andthe judgment of the prescribing physician.

Suitable quantities of DNA, e.g., plasmid or naked DNA can be about 1 μgto about 100 mg, preferably 0.1 to 10 mg, but lower levels such as 1-10μg can be employed. For example, an HIV DNA vaccine, e.g., naked DNA orpolynucleotide in an aqueous carrier, can be injected into tissue, e.g.,intramuscularly or intradermally, in amounts of from 10 μl per site toabout 1 nil per site. The concentration of polynucleotide in theformulation is usually from about 0.1 μg/ml to about 4 mg/mi.

The vaccine may be delivered in a physiologically compatible solutionsuch as sterile PBS in a volume of, e.g., one ml. The vaccines may alsobe lyophilized prior to delivery. As well known to those in the art, thedose may be proportional to weight.

The compositions included in the regimen descried herein for inducing animmune response can be administered alone, or can be co-administered orsequentially administered with other immunological, antigenic, vaccine,or therapeutic compositions.

Compositions that may also be administered with the vaccines includeother agents to potentiate or broaden the immune response, e.g., IL-15,IL-12, IL-2 or CD40 ligand, which can be administered at specifiedintervals of time, or continuously administered.

The vaccines can additionally be complexed with other components such aspeptides, polypeptides and carbohydrates for delivery. For example,expression vectors, nucleic acid vectors that are not contained within aviral particle, can be complexed to particles or beads that can beadministered to an individual, for example, using a vaccine gun.

Nucleic acid vaccines are administered by methods well known in the artas described in Donnelly et al. (Ann. Rep. Immunol. 15:617-648 (1997));Feigner et al. (U.S. Pat. No. 5,580,859, issued Dec. 3, 1996); Feigner(U.S. Pat. No. 5,703,055, issued Dec. 30, 1997); and Carson et al. (U.S.Pat. No. 5,679,647, issued Oct. 21, 1997), each of which is incorporatedherein by reference. One skilled in the art would know that the choiceof a pharmaceutically acceptable carrier, including a physiologicallyacceptable compound, depends, for example, on the route ofadministration of the expression vector.

As noted above, immunogenic DNA compositions can be delivered via avariety of routes. Typical delivery routes include parenteraladministration, intradermal, intramuscular or subcutaneous routes.Administration of expression vectors of the invention to muscle and byelectroporation can be a particularly effective method ofadministration, including intradermal and subcutaneous injections andtransdermal administration. Transdermal administration, such as byiontophoresis, is also an effective method to deliver expression vectorsof the invention to muscle. Epidermal administration of expressionvectors of the invention can also be employed. Epidermal administrationinvolves mechanically or chemically irritating the outermost layer ofepidermis to stimulate an immune response to the irritant (Carson etal., U.S. Pat. No. 5,679,647).

The immunogenic compositions can also be formulated for administrationvia the nasal passages. Formulations suitable for nasal administration,wherein the carrier is a solid, include a coarse powder having aparticle size, for example, in the range of about 10 to about 500microns which is administered in the manner in which snuff is taken,i.e., by rapid inhalation through the nasal passage from a container ofthe powder held close up to the nose. Suitable formulations wherein thecarrier is a liquid for administration as, for example, nasal spray,nasal drops, or by aerosol administration by nebulizer, include aqueousor oily solutions of the active ingredient. For further discussions ofnasal administration of AIDS-related vaccines, references are made tothe following patents, U.S. Pat. Nos. 5,846,978, 5,663,169, 5,578,597,5,502,060, 5,476,874, 5,413,999, 5,308,854, 5,192,668, and 5,187,074.

The vaccines can be incorporated, if desired, into liposomes,microspheres or other polymer matrices (see, e.g., Feigner et al., U.S.Pat. No. 5,703,055; Gregoriadis, Liposome Technology, Vols. I to III(2nd ed. 1993). Liposomes, for example, which consist of phospholipidsor other lipids, are nontoxic, physiologically acceptable andmetabolizable carriers that are relatively simple to make andadminister. Liposomes include emulsions, foams, micelles, insolublemonolayers, liquid crystals, phospholipid dispersions, lamellar layersand the like.

EXAMPLES ILLUSTRATING THE INVENTION Example 1 Core DNA VaccinationInduces Cross-Clade Specific Cellular Immune Responses in Mice

Results

Conserved Element DNA Vaccines

A set of conserved elements (CE) was identified in p24^(gag) composed ofamino acids (AA) highly conserved across the entire HIV-1 group M, asdetermined by using the Los Alamos HIV database (www site ishiv.lanl.gov/) [32]. A refined list of 7 CE was selected based onseveral criteria (FIG. 1A, see Materials and Methods): a minimum lengthof 8 AA; inclusion of specific epitopes that have been correlated withviral control (low viral loads) in vivo; and exclusion of epitopesassociated with high viral loads. The selected CE span 12-24 amino acidseach, and together a total of 124 AA, thus representing 54% of p24^(gag)sequence. The CE are highlighted on the p24^(gag) capsid ribbonstructure [97], revealing that they encompass most of the extendedcoiled regions of the p24^(gag) protein (FIG. 1B).

We generated multiple DNA-based vaccines in which the 7 CE werecollinearly arranged (FIG. 1C), and connected via short linker sequences(FIG. 1D), designed for efficient proteolytic cleavage [91, 92].Proteolytic processing of CE peptides in vitro revealed production ofoptimal epitopes or slightly extended optimal epitopes from 6 of the 7CE segments (S. Le Gall, in preparation). Therefore, the CE immunogen ispredicted to be able to present a significant number of native T cellepitopes after expression. For optimal arrangement of the differentsegments within the p24CE immunogens, the hydrophobicity of individualCE was taken into consideration (FIG. 1C). To avoid the stronglyhydrophobic N-terminus in the arrangement CE1-2-3-4-5-6-7 (left panel),which could impact the intracellular trafficking of the protein, the CE1peptide was placed at the C-terminus (right panel).

The majority of the AA included in the p24CE immunogens are essentiallyinvariant since they are found in >98% of HIV isolates. The length ofp24CE was expanded by including some less well-conserved AA (‘toggle’sites), thus expressing additional potentially immunogenic regions. Thisallowed the extension of the 7 CE to the length of 12-24 AA, asmentioned above, and led to two p24CE sequences differing by 7 AA, onein each CE (FIG. 1A), named p24CE1 and p24CE2. These two sequencescover >99% of all known HIV-1 group M sequences. The p24CE1 and p24CE2sequences were RNA/codon optimized [73-75] to maximize mRNA processing,transport, stability and translation (see Material and Methods). Thep24CE coding regions were cloned into the pCMVkan vaccine vector (p24CE;FIG. 1D). Additional expression plasmids were generated in order toalter the intracellular trafficking and processing of the p24CEproteins. Plasmids SP-p24CE1 and SP-p24CE2 contain the CM-CSF signalpeptide at the N-terminus of p24CE to promote secretion of the p24CEproteins. Plasmids MCP3-p24CE1 and MCP3-p24CE2 express fusion proteinswith the monocyte chemoattractant protein 3 (MCP-3) chemokine,previously shown to stabilize the encoded protein and to enhancetrafficking to antigen presenting cells [80,81. Plasmids LAMP-p24CE1 andLAMP-p24CE2 express fusion proteins with the human lysosomal associatedmembrane protein 1 (LAMP-1). Fusion of Gag to LAMP was previously shownto direct it to the lysosomal compartment and to facilitate access tothe MHC class II pathway as well as to the extracellular compartment[98-102].

Expression of the p24CE Proteins in Human Cells

The expression of the p24CE vectors shown in FIG. 1D was evaluated byWestern immunoblots using cell extracts and supernatants fromtransiently transfected HEK293 cells (FIG. 2). To control for equalloading, the membrane containing the cell-associated samples were probedwith an antibody against human beta actin as internal control (middlepanel) demonstrating that similar amounts of proteins were loaded intoeach lane which validates our conclusions regarding the stability anddifferent distribution of the proteins encoded by the differenttransfected plasmids (see below). Very low levels of the p24CE1 andp24CE2 proteins were detected in the cell-associated fractions (lanes 1and 5, respectively), and no proteins were found in the extracellularcompartment, indicating that the p24CE proteins were unstable. We alsonoted that p24CE2, differing only by 7 of the 124 AA from p24CE1,produced an even less stable antigen. The presence of the GM-CSF signalpeptide (SP) greatly increased the levels of both p24CE proteins (lanes2 and 6) in both the cell-associated and the extracellular fractions.These data indicate that the signal peptide altered the trafficking ofthe p24CE proteins, and promoted increase in stability and secretion. Wenoted the presence of additional bands of the secreted p24CE proteins,likely due to posttranslational modifications related to the alteredcellular trafficking (compare lane 2 and lane 1; lane 6 to lane 5). TheMCP3-p24CE (lanes 3 and 7) and LAMP-p24CE fusion proteins (lanes 4 and8) were also readily detectable, and thus these fusions greatlystabilized the p24CE proteins. MCPS-p24CE localization in theextracellular fraction (lanes 3 and 7) as several bands, was similar toour previous report on a MCPS-Gag fusion protein [81]. The LAMP-p24CEproteins accumulated primarily in the cell-associated fraction (lanes 4and 8), although some protein could also be found in the extracellularfraction, as we previously observed for the LAMP-p55^(gag) protein[101]. These data shoved that altering the trafficking of the p24CEproteins, by adding the GM-CSF signal peptide or upon fusion to the MCP3or LAMP molecules, enhanced the stability and modulated the traffickingof p24CE proteins.

Vaccination with p24CE Induces GE-Specific Cellular Immune Responses inC57BL/6 Mice

We next evaluated the immunogenicity of different p24CE proteins afterDNA vaccination of C57BL/6 mice. Groups of mice (N=5) were vaccinatedtwice (0 and 4 weeks) with the indicated p24CE plasmids or sham plasmidDNA, as negative control, by intramuscular injection followed by in vivoelectroporation (EP). Two weeks after the last vaccination (week 6), themice were sacrificed and the presence of CE-specific cellular responseswas determined by polychromatic flow cytometry. Splenocytes from theindividual animals from each of the vaccine groups and the sham DNAinoculated negative control group were stimulated with a Group Mconsensus Gag peptide pool (15-mer peptides overlapping by 11 AA) (FIG.3A) or with a COT-M peptide pool (10-mer overlapping by 9 AA) consistingof both p24CE1 and p24CE2 sequences (FIG. 3B). The use of the 15-merpeptide pool allowed for the detection of both CD4⁺ and CD8⁺ T cellresponses, whereas the 10-mer peptide pool favors mainly CD8⁺ T cellresponses. Vaccination with plasmids expressing p24CE or the secretedp24CE (SP-p24CE) proteins induced both CE-specific CD4⁺ and CD8⁺ T cellimmune responses (FIG. 3A) contrast, vaccination with the p24CE fusionproteins, MCP3-p24CE or LAMP-p24CE, elicited CE-specific responses thatwere almost exclusively mediated by CD4⁺ T cells. In agreement withthese results, splenocyte stimulation with 10-mer peptide pools, whichare mainly associated with MHC class I antigens, induced very lowresponses in MCP3-p24CE DNA vaccinated mice and no responses in theLAMP-p24CE immunized mice, which verified the previous conclusions (FIG.3B). We hypothesize that altered intracellular trafficking of the p24CEfusion antigens could be responsible for the distinct preference forCD4⁺ or CD8⁺ T cell responses. Responses elicited by p24CE1 proteinswere in general higher than those induced by p24CE2 (FIGS. 3A and 3B,note the different scales for p24CE1 and p24CE2), likely reflecting thehigher expression of p24CE as indicated by the transient transfectionexperiments (see FIG. 2). As expected, no cellular responses were foundin splenocytes from sham DNA vaccinated mice.

The cross-reactivity of the induced responses was analyzed using peptidepools representing different HIV-1 chides (A, B, and C; see also FIG.1A). SP-p24CE1 DNA vaccination induced cross-clade reactive CD4⁺ andCD8⁺ cellular responses, which were similar in magnitude to thoseobtained with the Group M peptide pool (FIG. 3C). Cross-Clade reactivitywas also obtained upon vaccination with the other p24CE plasmids (datanot shown). In contrast, splenocytes from mice immunized with sham DNAfailed to recognize peptides from any of the three glade-specificpeptide pools.

Fine Specificity of CE-Specific T Cell Responses from Vaccinated C57BL/6mice

Next, we assessed the distribution of the p24CE-induced cellularresponses among the different CE (FIG. 4). Pooled splenocytes from theDNA vaccinated C57BL/6 mice (N=5/group) were stimulated with Group Mconsensus peptide pools (15-mer) encompassing the 7 individual CE.Polychromatic flow cytometry was used to determine the frequency of theCE-specific IFN-γ producing T cells and to discriminate between CD4⁺ andCD8⁺ T cell responses. Immunization with the different p24CE1 (leftpanels) and p24CE2 (right panels) plasmid DNAs induced cellularresponses to CE1 and CE6, which were mediated almost exclusively by CD4⁺T cells. Interestingly, mice immunized with plasmids encoding the nativep24CE: protein (p24CE and SP-p24CE) developed also high CD8⁺ mediatedcellular responses to CE2. These data are in agreement with the cellularlocalization of the encoded proteins: the native p24CE protein remainsmainly intracellular, while the SP-p24CE and MCP3-p24CE fusion areactively secreted and the LAMP-p24CE associates with the MHC class TIcompartment. Low levels of CD8⁺ T cell responses to CE3 were alsoidentified upon immunization with the p24CE and the MCP3-p24CE plasmids.In conclusion, the p24CE proteins induced responses to 4 of the 7 CE(CE1, CE2, CE3, CE6) in mice, although these responses were generallylower in animals immunized with the p24CE2 plasmids, demonstrating thatvaccination induced broad CD4⁺ and CD8⁺ T cell responses.

p24CE Induces Broader Immune Responses than the Full-Length p55^(gag)

We compared the immune responses to the individual CE upon vaccinationwith a p55^(gag) plasmid DNA or with a mixture of SP-p24CE1 andSP-p24CE2 DNAs. The mice (N=0.5 group) received 3 vaccinations (week 0,3 and 6) and were sacrificed at week 8 (FIG. 5A). Vaccine-induced T cellresponses were analyzed from pooled splenocytes (FIG. 5B) stimulatedwith 15-mer peptide pools specific for p24^(gag) of clade A, B, or C(left panel) and the group M consensus (right panel). The overallresponses induced by the p55^(gag) immunogen were lower than thoseobtained by the p24CE immunogen, and remarkably, lacked CD8⁺-specific Tcells. Using peptide pools spanning the individual CE (FIG. 5C) showedthat p55^(gag) DNA elicited low responses to CE1 and CE6 only (rightpanel), mediated exclusively by CD4⁺ T cells. In contrast, vaccinationwith SP-p24CE DNA mixture elicited higher responses towards several CE(CE1, CE2 CE3 and CE6), as also expected from the data shown in FIG. 4.

We also evaluated the quality of the cellular immune responses elicitedby the different immunogens (FIG. 5D) using the p24^(gag) peptide poolfollowed by intracellular cytokine staining and polychromatic flowcytometry. Vaccination with p55^(gag) DNA induced primarily CD4⁺ (red) Tcell responses, while p24CE vaccination induced both CD4⁺ (red) (CE1 andCE6) and CD8⁺ (black) (CE2 and CE3) T cell responses (FIGS. 5B and 5C,top panel). Both immunogens induced effector memory T cells (CD44^(hi)an and CD62L^(neg)) (FIG. 5C, middle panel), which were mainly CD4⁺(0.28% of total T cells) in mice vaccinated with p55^(gag) DNA, and bothCD4⁺ (0.72% of total T cells) and CD8⁺ (0.37% of total T cells) in themice vaccinated with the SP-p24CE DNA. Further analyses revealed thatantigen-specific IFN-γ⁺ T cells produced TNF-α and expressed CD107a onthe surface upon stimulation with antigen, indicating induction ofcytotoxic T cells (FIG. 5D, bottom panel). We also noted that the CD8⁺ Tcells (black), induced only by SP-p24CE DNA vaccination, expressedhigher levels of CD107a and lower levels of TNFα than the CD4⁺ T cells(red), a phenotype consistent with the degranulation associated with CTLactivity. Collectively our results show that the p24CE vaccine increasedbreadth and magnitude of cellular responses to p24^(gag) region in DNAvaccinated mice, by inducing robust responses to several of the highlyconserved elements, and that the responses are multifunctional, adesired feature for an effective HIV vaccine,

Vaccination with p24CE Induces Cross-Clade Reactive Humoral ImmuneResponses

We next examined the induction of humoral immune responses using pooledplasma samples from p24CE DNA vaccinated mice (N=5/group) by an ELISAmeasuring clade B p24^(gag) responses (FIG. 6A). The different p24CEantigens readily induced high levels of humoral responses with titerssimilar or greater than those achieved in p55^(gag) DNA vaccinated mice(except p24CE2, middle panel). Vaccination with p24CE DNA inducedantibodies to both the p24CE proteins (FIG. 6B, top panel, lanes 2 and3) as well as to the processed p24^(gag) protein (lane 1). In contrast,the antibodies induced by p55^(gag) DNA vaccination readily detectedp24^(gag) (FIG. 6B, bottom panel, lane 1), but failed to recognize thep24CE proteins (lanes 2 and 3). Thus, similar to the cellular immuneresponses (see FIG. 5), the antibodies elicited upon vaccination withfull-length p55^(gag) DNA were unable to recognize the conservedelements.

We also examined the cross-clade reactivity of these responses byWestern immunoblot analysis (FIG. 6C). Membranes containing p55^(gag)proteins from consensus clades A and C, clade B (HXB2) and COT-M,obtained from transiently transfected were probed with pooled plasmasamples from mice vaccinated with plasmids expressing SP-p24CE1,SP-p24CE2 or p55^(gag). The Western immunoblot assays showed that theantibodies induced by the p24CE and p55^(gag) DNA vaccinated mice detectthe different p55^(gag) proteins. These data suggest that similar top55^(gag), p24 CE vaccinated mice induce cross-clade reactiveantibodies.

Together, these data show that p24CE DNA vaccination induced strongImmoral (FIG. 6) and cellular (FIG. 4) immune responses to the highlyconserved elements in p24^(gag), and that CE segments are not or onlypoorly immunogenic when expressed as part of the complete p55^(gag) inDNA vaccinated C57BL/6 mice.

Discussion

The experiments performed in mice demonstrated that a DNA vaccineexpressing 7 selected highly Conserved Elements within HIV-1 p24^(gag)can be produced and that this DNA vaccine is immunogenic in comparisonto DNA-encoded full-length native p55^(gag). We have previouslydemonstrated that individuals chronically infected with HIV-1 developcellular immune responses specific for the peptides encoded by the 7conserved elements described in this work [34]. Furthermore, we foundthat the breadth, magnitude and avidity of these cellular responses tosome CE were significantly higher among patients able to control HIV-1infection, which suggests that responses against these conserved regionsare clinically relevant [34].

Starting from our understanding of the rules for robust gene expression,and to avoid the escape potential of HIV, we constructed optimized DNAvectors that express maximal levels of new artificial immunogens basedon highly conserved elements of the p24^(gag) region. This vaccinedesign is based on two principles, (i) the immunogen must includecritical and highly conserved elements of the virus that cannot mutatewithout a severe loss in viability, and (ii) the immunogen must excludeHIV epitopes that are capable of mutating without significantlyaffecting viral fitness. The former may induce responses against a largenumber of HIV isolates, and the latter avoids immunodominant competitionfrom variable regions, which may render ineffective the vaccine-inducedimmune response. Not only expression, but also the stability andpresentation of the artificial antigens encoded by the DNA vectors wereoptimized To this end, different fusion constructs were designed. Wepreviously noted that either addition of a signal peptide or fusion toeither MCP3 or LAMP were beneficial for protein expression [81,80,94],and found that these modifications also stabilize the p24CE proteins.

To maximize stimulation of CD4⁺ cells in addition to CD8⁺ T cells, wedesigned S secreted immunogens. The p24CE antigen linked to the signalpeptide of GM-CSF was expressed at high levels and also produced bothCD4⁺ and CD8⁺ antigen-specific T cells as well as good antibody titers.In contrast, p24CE proteins fused to MCP-3 or LAMP directed thedevelopment of mostly CD4⁺ T cell responses. These studies show that itis possible to manipulate many properties of an antigen, altering theimmune response in predictable ways. Although CD8⁺ T cell responses havebeen linked to control of viremia, we have also reported that cytotoxicCD4⁺ T cell responses contribute to viral control [103]. The p24CEvaccine induced both CD4⁺ (CE1 and CE6) as well as CD8⁺ (CE2 and CE3)specific T cell responses in the C57BL/6 mice; these CD8⁺ T cells hadthe functional phenotype of mature CTLs and were absent in miceimmunized with the DNA encoding p55^(gag). The molecules generated allowthe selection of the most optimal combinations to achieve the bestprotective response for HIV prophylaxis. We have found that p24CEproteins are more immunogenic than the full-length Gag protein,expanding the quality of cellular responses to recruit CD8⁺ T cells withthe functional properties of canonical CTLs in C57BL/6 mice. Thesefindings suggest that the peptides containing the CE regions producedfrom the full-length p55^(gag) antigen were not recognized efficientlyby the T cells. This could be due to poor antigen processing orpresentation, or alternatively due to interference by immunodominantpeptides from other regions within p55^(gag), which are able to divertor inhibit immune response. In addition, as shown in FIGS. 3 and 4, thetrafficking of the protein greatly affected its immunogenicity, with thep24CE and SP-p24CE eliciting the highest and most balanced CD4 and CD8responses. Selection of the most optimal p24CE protein induced higherand broader immunogenicity than p55^(gag). We have recently also shownthat dendritic cells in vitro loaded with RNA encoding the p24CEdescribed in this work are able to stimulate T cell responses when mixedwith autologous PBMC from HIV patients or to induce de novo T cellresponses in PBMC from healthy donors. The responses elicited by p24CEwere usually as high as those by full-length Gag [33].

All the immunogenicity studies described in the present work wereperformed in C57BL/6 mice. In our experience, p55^(gag) DNA immunizationusing the Balb/c mouse mode induces higher T cell responses, but thoseresponses are almost exclusively directed towards a singleimmunodominant epitope, AMQMLKETI, which is present in our p24CEconstruct. Therefore, to avoid the restrictions imposed by this limitedrepertoire, we chose the C57BL/6 mouse model for the work describedherein. We found very low primary immune responses to the CE regionsafter DNA vaccination using full-length p55^(gag). It will be ofinterest to further examine whether other vaccine modalities, i.e.recombinant viral vectors, expressing p55^(gag) are able to inducehigher immune responses to the CE. To our knowledge, this study is thefirst comparative evaluation of immunity induced by a full-lengthimmunogen and that induced by highly conserved elements from within thesame protein. Our analysis points to the negative effect of regionsoutside of the defined conserved elements, which, it is important tonote, are present in the full-length wild type as well as in theconsensus and mosaic molecules as well as in the reported epitopeimmunogens. Thus, the use of the highly Conserved. Element platformoffers the advantage of focusing the immune responses to the invariableepitopes present in the viral proteome. Similar to the work describedhere, Letourneau et al. [11] previously demonstrated that a chimericprotein containing a string of several invariable regions from the HIV-1proteome was immunogenic, but a direct comparison with the samesequences expressed within the natural proteins was not performed. Inour study, we applied more stringent criteria to define conservedelements resulting in shorter peptide sequences (12-24 AA) that excludeadjacent more variable segments. In addition, our analysis of the immuneresponses was performed using peptide pools strictly confined to theconserved segments defined as immunogens and, therefore, thecontribution to the T cell responses of putative artificial new epitopescreated by the boundaries was completely excluded. In conclusion, weshowed that the p24CE DNA vaccine induced broad cross-clade reactivecellular and humoral responses in vaccinated mice. We detected robustimmune responses, including CD8⁺ T cells, to several CE upon p24CE DNAvaccination in mice, whereas only very poor (CD4⁺ only) or no responsesto the CE were obtained by DNA vaccination with vectors expressingfull-length p55^(gag). Thus, the inclusion of DNA vectors expressing theconserved elements is a promising vaccine strategy to induce broaderimmunity compared to vaccination with the p55^(gag) DNA alone. Theseresults suggest further evaluation of the p24CE antigens in macaques.

Materials and Methods

p24^(gag) Conserved Elements Selection

Using all HIV-1 M group p24^(gag) coding sequences available in the 2009Los Alamos database, we identified sequences of at least 8 AA in length,in which all AA were conserved in at least 98% of all sequences. Thisrequirement was then relaxed in two ways: First, using available datathat correlated epitope recognition with clinical viral load, we soughtto include complete epitopes that were associated with loin viral loadand exclude epitopes that were associated with high viral load.Secondly, we allowed 1 toggle (variable) site/CE segment if the 2 mostcommon AA at that site are together found in >99% of all known sequences[32]. To accommodate this variation, we created two plasmids, each with7 CE segments from 12-24 AA in length, separated by 2-4 AA spacers(typically Ala-Ala-X) and differing only by the single toggle AA. Thelength and sequence of the spacers was set based on the existingknowledge of cleavage specificities and peptide availability [104], aswell as to avoid fortuitous junctional homologies to HIV and the humanproteome, the latter determined by searching against the HIV and humanprotein sequence databases.

DNA Plasmids

The p24CE and gag gene coding sequences were designed by RNA/codonoptimization for efficient expression in mammalian cells [73-75] andchemically synthesized (GeneArt, Life Technologies, Grand Island, N.Y.).The genes were cloned into the pCMVkan vector [81] optimized for highgene expression. pCMVkan contains the human cytomegalovirus promoter,and the expressed transcripts contain a optimal surrounding for the AUGinitiator codon from HIV-1 tat that prevents initiation of translationfrom internal AUGs [105], the bovine growth hormone (BGH)polyadenylation site, and the kanamycin resistance gene. This vectordoes not contain any splice sites or introns. The p24CE1 and p24CE2proteins were produced from independent vectors (plasmids 164H and 182H,respectively). The secreted forms SP-p24CE1 and SP-p24CE2 contain theGM-CSF signal peptide (AA 1-17; Genbank accession Nr. NP_000749) at theN terminus (plasmids 234H and 235H). The MCP3-p24CE1 and MCP3-p24CE2(plasmids 167H and 201H) are fusion proteins with the monocytechemoattractant protein 3 (MCP-3) [80-81]. The LAMP-p24CE1 andLAMP-p24CE2 (plasmids 191H and 202H) are fusion proteins with thelysosomal associated membrane protein 1 (LAMP-1) [98-101]. Full-lengthp55^(gag) proteins were produced from RNA/codon optimized genes clonedinto the pCMVkan plasmid, expressing Gag from clade A (plasmid 187H,Genbank accession number AAQ98129), clade B (plasmid 114H, HXB2, Genbankaccession number AAB50258), clade C (plasmid 160H, Genbank accessionnumber AAD12096) and the center-of-tree COT-M (222H) [106]. Forimmunizations HXB2 p55^(gag) was used. Endotoxin-free DNAs were preparedusing Qiagen kit according to the manufacturer's protocol (Qiagen,Valencia, Calif.)

Transfection and Protein Analysis

DNA plasmids were transfected into 1×10⁶ HEK-293 cells using the calciumphosphate co-precipitation technique. Culture supernatants and cellswere harvested 24 or 48 hours later, and protein expression wasvisualized by Western immunoblot analysis. The proteins were resolved on10% or 12% NuPAGE Bis-Tris gels (Invitrogen, Carlsbad, Calif.),transferred onto nitrocellulose membranes (Invitrogen), which wereprobed with a goat anti-p24^(gag) antibody (dilution 1:3000, provided byL. Arthur, SAIC, NCI, Frederick) followed by anti-goat IgG-HRP labeledantibody (dilution 1:10,000; Calbiochem, EMD chemicals, Gibbstown, N.J.)or with plasma (1:200 dilution) from DNA vaccinated mice followed byanti-mouse IgG-HRP labeled (1:10,000 dilution, GE Healthcare,Piscataway, N.J.). As control, the membranes were probed with anti-humanpan-actin antibody (clone C4, EMD Millipore, Billerica, Mass.) at adilution of 1:10,000. The bands were visualized using the enhancedchemiluminescence (ECL) plus Western blotting detection system (GEHealthCare, Piscataway, N.J.).

Mouse DATA Vaccination Studies

Female C57BL/6N (6 to 8 weeks old) were obtained from Charles RiverLaboratories, Inc. (Frederick, Md.) and were housed at the NationalCancer institute, Frederick, Md., in a temperature-controlled,light-cycled facility. The mice were immunized with 20 μg of the vaccineDNAs by intramuscular injection followed by in vivo electroporation byELGEN® constant current electroporation device (Inovio Pharmaceuticals,Inc, Blue Bell, Pa.). As negative controls, a group of mice receivedequal amount of sham DNA following the same immunization protocol. Theanimals were vaccinated two (0 and 4 weeks) or three times (0, 3, and 6weeks), and were sacrificed 2 weeks after the last vaccination whenspleens and blood were collected for the analysis of cellular andhumoral responses.

Intracellular Cytokine Staining

The frequency of antigen specific cytokine⁺ T cells was measured usingpolychromatic flow cytometry, as previously described [80]. Thefollowing set of 15-mer Gag peptide pools, overlapping by 11 AA, wereused to stimulate the vaccine-induced cellular responses: HINT-1consensus clade A (Cat#8116), consensus clade C (Cat#8118), and Group MConsensus (Cat#11057), obtained from the AIDS Research and ReferenceReagent Program (Germantown, Md.); Gag 15-mer from HXB2/Clade B(Infinity Biotech Research & Resource, Inc, Aston, Pa.). Peptide poolsspanning p55^(gag), p24^(gag) or only CE were generated. In addition, weused pools of 10-mer peptides overlapping by 9 AA from COT-M spanningthe individual CE1-CE7, not including linker sequences, of p24CE1 andp24CE2 (peptide synthesis facility of the Massachusetts GeneralHospital, Boston). Splenocytes were cultured at 37° C. and 5% CO₂ at adensity of 2×10⁶ cells/ad in complete RPM1-1640 medium containing Gagpeptide pools at a final concentration of 1 μg/nil of each peptide. Inall experiments, splenocytes cultured in medium without peptide pools orstimulated with phorbol myristate acetate (PMA) and calcium ionophore(Sigma, St. Louis, Mo.) were used as negative and positive controlrespectively. Protein secretion was blocked by the addition of monensin(GolgiStop, BD Biosciences) 1 hour after stimulation. After 12 hoursincubation, the cells were harvested and cell surface staining wasperformed using the following antibody cocktail: CD3-APCCy7, CD4-PerCP,and CD8-Pacific Blue (BD Pharmingen, San Diego, Calif.). Splenocyteswere washed twice, fixed, permeabilized with Cytofix/Cytoperm (BDPharmingen) and staining for intracellular cytokine detection wasperformed using IFN-γ-FITC (BD Pharmingen). In another set ofexperiments, the antibody cocktail for surface staining included:CD3-AF700, CD4-PerCP, CD8-Pacific Blue, CD44-V500, CD62L-PE CD107a-PECy7 (BD Pharmingen). The anti-CD 107a antibody was added during theculturing of splenocytes with peptides. IFN-γ-APC and TNF-α-APC Cy7 (BDPharmingen) were used for intracellular cytokine staining. Afterintracellular staining, the cells were washed twice and the samples wereanalyzed on an LSR II flow cytometer (BD Pharmingen). Data analysis wasperformed using the FlowJo platform (Tree Star, Inc., Ashland, Oreg.).All antigen specific responses are reported after subtracting valuesobtained from the samples without peptide stimulation. Only splenocytesgiving a response more than two fold higher than the value of the samplewithout peptides (medium alone) were considered positive.

Antibody Assays

Serial dilutions of plasma samples were analyzed by standard HIV-1 cladeB p24^(gag) ELISA (Advanced Bioscience Lab, Rockville, Md.), measuringoptical absorbance at 450 nm.

Example 2 Core DNA Vaccination Induces Cross-Clade Specific CellularImmune Responses in Macaques

Results

Vaccination with Gag DNA Induces Poor p24^(gag) Conserved Element(CE)-Specific Cellular Immune Responses in Macaques

We first investigated whether vaccination of macaques with a plasmidexpressing p55^(gag) was able to elicit immune responses to the 7 CE[32,34] identified within the p24^(gag) sequence (FIG. 7). Four animalswere vaccinated twice (0, 2 month) with COT-M p55^(gag) DNA by IMinjection followed by in vivo electroporation (EP). Seven animalspreviously vaccinated with a plasmid expressing the full-lengthp55^(gag) delivered intramuscularly (N=4) [82] or with a plasmidexpressing the p37^(gag) protein delivered via IM/EP (N=3) [67] wereincluded in the analysis. Induction of Gag-specific responses wasevaluated upon stimulation of PBMC with a p24^(gag) specific peptidepool as well as with a CE-specific peptide pool (see Material andMethods). All 11 macaques developed readily detectable responses to thep24^(gag) region, however only 5 of the 11 macaques (45%) developedresponses to CE (FIG. 7B). Next, we analyzed the specificity of theresponses towards the individual conserved elements in the 5 macaquesthat showed CE recognition (FIG. 7C). We found that 3 macaques (L985,P574, R288) recognized only 1 CE (CE3 or CE5), whereas 2 macaques (R067and M121) developed responses to 2 CE (CE4, CE5 and CE5, CE6,respectively). Moreover, only animal L985 developed significant CD8⁺ Tcell responses against the CE, while the other 4 animals showed almostexclusively CD4⁺ T cell mediated responses. These CE-specific T cellresponses included CD4⁺ and CD8⁺ T cells with cytotoxic potential, asjudged by the presence of antigen-specific Granzyme B⁺ T cells in all 5animals (FIG. 7D).

The lack of CE recognition in most of the vaccinated animals raised theconcern drat the immunogenicity of the CE epitopes within the Gagprotein may be impaired due to either suboptimal processing andpresentation of the CE-containing peptides, or immunodominance exertedby variable regions within Gag directing the CTL responses away from CE.

Vaccination with p24CE DNA Induces Cellular Immune Responses in Macaques

To test whether broader immune responses to the CE could be elicited inmacaques, we vaccinated animals with a mixture of the two p24CE DNAvectors which were engineered to express the 7 CE collinearly arranged.(See Example 1). The two proteins differed by 1 AA (‘toggle’) per CE,(SP-p24CE1 and SP-p24CE2) (FIG. 7A). Four animals were vaccinated twice(0, 2 month) with p24CE DNA using IM/EP delivery. Cellular immuneresponses were measured in blood samples collected 2 weeks after the2^(nd) vaccination (EP2wk2). Two additional macaques (M437, P314),previously immunized with p24CE plasmids, were also included in thisanalysis. All 6 macaques developed CE-specific cellular responses (FIG.8A), as measured by the production of IFN-γ with a frequency rangingfrom 0.1% to 0.6% of total T cells. The overall levels of responses werenon-significantly lower compared to those of the p55^(gag) DNAvaccinated animals data not shown). These responses included both CD4⁺and CD8⁺ T cells, although the CD8⁺ T cell responses were dominant in 4of the 6 vaccinated animals (FIG. 8A). These results are in contrast tothose obtained upon p55^(gag) DNA vaccination, where only 5 out 11immunized animals developed CE responses, mediated mainly by CD4⁺ Tcells (FIG. 7). Mapping of the CE-specific responses (Figure BB)revealed recognition of all CE except CE1 and CE7, using out-bredanimals with different MHC class I haplotypes. Comparison of animalsvaccinated with p24CE or Gag DNA shows that there was no apparentcorrelation between haplotype and ability to develop responses to CE,hence the differences could be attributed to the immunogen. Five of the6 CE vaccinated macaques developed responses to 3 CE and only one animal(M437) showed responses to 1 CE. Phenotypic analysis of theantigen-specific T cells revealed both central (CD28⁺CD95⁺) and effectormemory (CD28⁻CD95⁺) (FIG. 8C, top panels). A subset of the CE-specificIFN-γ⁺ T cells also expressed granzyme B, indicating a cytotoxicphenotype (Figure BC, bottom panels), thus eliciting cytotoxic.CE-specific responses. These results indicate that, similar to ourobservation from vaccinated mice (Example 1), the CE DNA vectors areimmunogenic in all 6 macaques and that most (5 of 7) of the CE wereimmunogenic. These data also demonstrate that CE-containing peptides areprocessed and presented properly and suggest that the failure to induceCE-specific responses from p55^(gag) or p37^(gag) is likely the resultof immunodominance exerted by epitopes located in the variable regions,

Vaccination with p24CE DNA Induces Broader and Higher Levels ofPolyfunctional CE-Specific T Cell Response than Vaccination withp55^(gag) DNA

We further dissected CE immunogenicity by comparing the cellularresponses induced by p24CE and p55^(gag) DNA vaccination. First, wecompared the number of CE recognized in the macaques vaccinated with DNAexpressing the p24CE (N=6) Of full-length p55^(gag) or p37^(gag) (N=11)(FIG. 9A), immunization with p24CE induced responses to significantlymore CE (p=0.0006; range 1-3 CE, median 3) than Gag DNA vaccination(range 0-2 CE) (FIG. 9A). These data demonstrate that p24CE DNA inducedresponses to more CE, indicating increased breadth of responses comparedto p55^(gag) DNA vaccination.

We also compared polyfunctionality (production of IFN-γ, TNF-α, CD107aand granzyme B) of the T cell responses upon stimulation withCE-specific peptides. FIG. 9B shows the distribution of CE-specificpolyfunctional T cells from representative macaques that received eitherp24CE DNA (top panel) or p55^(gag) DNA (middle panel). The proportion ofpolyfunctional T cells (1- to 4-function) is also shown as pie charts(right panels). These results demonstrate that p24CE DNA vaccinationelicited higher CE-specific cytotoxic T cell levels than p55^(gag) DNAvaccination. The frequency of CE-specific T cells secreting twocytokines, expressing granzyme B and able to degranulate upon antigenrecognition (4-function) was also significantly higher (p=0.03) inmacaques immunized with p24CE DNA (bottom panel). Together, these datashow that the p24CE immunogen elicited significantly higher responses,including to more CE, and that these responses are multifunctional andhave cytotoxic properties.

P55^(gag) DNA Vaccination Boosts Pre-Existing CE-Specific T CellResponses

Given that repeated vaccination with p55^(gag) DNA failed or only poorlyinduced de nova CE-specific T cell responses (FIG. 7), we investigatedwhether full-length gag DNA vaccination could boost and/or broadenpre-existing CE-specific immunity. The p24CE-vaccinated macaquesreceived an additional vaccination with a plasmid expressing COT-Mp55^(gag) DNA (FIG. 10A; group 1). This led to a significant increase(p=0.002) in CE-specific responses, reaching in some animals more than1-2% of the total T cell population (FIG. 10B). Analysis of thepolyfunctionality of these responses showed that the frequency ofCE-specific T cells with 4 functions was also significantly boosted(p=0.002; FIG. 10C). Boosting with p55^(gag) DNA also induced de novoresponses to p17^(gag) and C-terminal regions of Gag, thereby increasingthe total Gag responses to levels similar to those obtained with thegag/p24CE DNA vaccine. Additionally, virtually the complete set ofpre-existing responses to individual CE was boosted in 6 macaques (FIG.11A, left panel). The number of the CE found to be immunogenic uponp24CE vaccination (1-3 CE/animal) increased to 2-4 CE/animal upon GagDNA boost. These findings confirmed that the suboptimal responsesinduced by priming with full-length Gag were not related to the absenceof processing or presentation of CE-containing peptides, but rather totheir inability to induce de novo responses in the presence of other,likely more dominant, Gag epitopes outside of CE. Thus, theimmunodominance exerted by Gag epitopes outside of CE was lost in thepresence of pre-existing CE-specific responses.

We also investigated whether p24CE DNA vaccination could alter theCE-specific immunity in macaques previously vaccinated with p55^(gag)DNA (FIG. 10A; group 2). Vaccination with p24CE DNA minimally increasedthe pre-existing CE-specific responses in 3 out of 4 macaques, (FIG.10B, group 2) and modestly increased the polyfunctional CE responses intwo of the vaccinated animals (FIG. 10C, group 2), although thisincrease was not statistically significant. Analysis of the individualCE (FIG. 11B) showed no new CE responses upon p24CE boost. Theheterologous p24CE DNA boost did not alter the pre-existing CD4⁺ or CD8⁺T cell distribution. Thus, the immunodominance exerted by epitopesoutside of CE could not be overcome by p24CE vaccination, as thisvaccine regimen did not alter the magnitude or breadth of Gag responses.

P24CE DNA Vaccination Induces Humoral Immune Responses that Recognizep24^(gag)

The development of Gag-specific humoral immune responses was alsomonitored over the course of study (FIG. 12A) using a p24^(gag) ELISA.Upon vaccination with p24CE DNAs (group 1) antibodies recognizingp24^(gag) were readily delectable and peaked 2 weeks after EP2 (meanreciprocal end-point dilution titer 5.2 log). Similarly, the p24^(gag)antibody titers upon COT-M p55^(gag) DNA vaccination also peaked 2 weekspost EP2 (mean reciprocal end-point dilution titer 5.3 log). Thus, bothvaccines elicited similar p24^(gag) antibody titers.

We further assessed the ability of these antibodies to recognize thep24CE proteins as well as processed p24^(gag) by Western immunoblots.The data from two representative macaques from each group are shown(group 1: L862 and M166, and group 2: P574 and R288) with similar dataobtained from all animals from both vaccine groups. Plasma from macaquesvaccinated with p24CE (Group 1) recognized naturally processed p24^(gag)produced from a chide B molecular clone of HIV-1 (FIG. 12B, lane 1) aswell as the p24CE1 (lane 2) and p24CE2 (lane 3) proteins. In contrast,vaccination with p55^(gag) DNA (group 2) induced antibodies thatstrongly react with p24^(gag) (lane 1), but failed to recognize p24CEproteins (FIG. 12B lanes 2 and 3). We conclude that only the p24CE DNAvaccination induces robust cellular and humoral responses to theconserved elements.

Lastly, we tested whether boosting with the heterologous DNA (EP3)affected the pre-existing humoral immune responses. ELISA assays showedsimilar increase in p24^(gag) antibody levels in both groups (FIG. 12A).All Western immunoblot assays (FIG. 12B) were performed in parallelusing the same plasma sample dilution and the same exposure time of themembrane to allow comparison of before and after the respective boosts.Following p55^(gag) DNA boost of the p24CE DNA vaccinated animals (group1), stronger reactivities to both p24^(gag) (lane 4) as well as p24CEproteins (lanes 5 and 6) were found. These data demonstrate thatp55^(gag) DNA vaccination was able to substantially boost the CE-primedhumoral immune responses despite its failure to induce de nova antibodyresponses able to recognize the CE protein. Vaccination of the p55^(gag)DNA primed animals with p24CE DNA (group 2, bottom panels) showedinduction of antibodies to p24CE proteins (lanes 5 and 6) and increasedreactivity to p24^(gag) (lane 4). Note, a significantly higher amount ofplasma was used in order to detect p24CE proteins from the animals ingroup (dilution 1:500) compared to group 1 (dilution 1:2000). Thus,these data indicate that the heterologous p24CE DNA boost induced lowlevel CE-specific responses, rather than inducing anamnestic responses(group 2). Together, these data show that prime immunization with p24CEDNA can alter the immunodominance of both cellular and humoral immuneresponses and that the immunodominance of epitopes outside of CE is notovercome by boosting with CE if the animal's vaccination involvedpriming with p55^(gag). Therefore, priming with p24CE DNA followed bythe heterologous p55^(gag) DNA boost is a preferred approach to achievebroad and high cellular and humoral immune responses to the highlyconserved elements of HIV-1 p24^(gag) protein.

Discussion

We have described DNA vectors encoding collinearly 7 highly conservedelements of the HIV-1 Group M p24^(gag) protein, and we have reportedthat vaccination of mice with these DNAs induced both cellular andhumoral responses [57]. In the current report, we demonstrated thatvaccination of rhesus macaques with these DNA vectors inducedCE-specific cellular and humoral immune responses. Detailed analysis ofcellular immune responses showed that p24CE DNA vaccination inducedcytotoxic CD4⁺ and CD8⁺ T cells against CE and that the elicited T cellresponses were polyfunctional. Therefore, our conserved element DNAvectors show desired features for an effective vaccine. Our vaccineregimen also shows a promising approach to overcoming a problem in theHIV vaccine field, where attempts to induce both antigen-specific CD4⁺and CD8⁺ T cell responses and to broaden the vaccine-induced immunity toinclude subdominant epitopes have been less successful, even with areported EP DNA/Ad boost immunization strategy [83].

Importantly, we found that the p55^(gag) vaccine elicits no or only poorresponses to CE. We also analyzed the responses from a previous report[4], where macaques were vaccinated with consensus or mosaic p55^(gag)DNA as prime followed by recombinant Adenovirus boost. We found that 5of the 12 animals that received the consensus molecule and 6 of 12 thatreceived the mosaic molecules developed CE responses ranging from of 0-2(consensus) and of 0-4 (mosaic) CE responses/animal, whereas severalepitopes outside the CE were immunogenic in all the animals. In thestudy reported herein, we found that 5 of 11 macaques vaccinated withfull-length COT-M or HXB2 p55^(gag) DNA developed CE-specific responses(0-2 CE/animal), whereas epitopes outside the CE were immunogenic in allthe macaques. The data of the two studies are thus in good agreement,although the methods of analysis were not identical [peptide mapping [4]versus analysis with CE-specific mixture of 15-mer and 10-mer peptides(this report)]. Irrespective of the nature of Gag vaccine (consensus,mosaic or wild type), we found responses to CE in only 42-50% of theanimals, and the responses were to very few CE/animal, suggesting thatimmunodominant epitopes within Gag focus the CTL response away fromthese conserved targets. In this report, we experimentally tested thishypothesis and demonstrated that immunodominance of variable regions isindeed responsible for the poor immunogenicity of the CE.

Although vaccination with either p55^(gag) or p37^(gag) induced stronghumoral responses, we found that these antibodies fail to cross-reactwith the CE protein, in contrast, our engineered p24CE DNA vaccinereadily induced both antibodies and cell-mediated responses to severalCE, bypassing the restriction associated with full-length Gagvaccination. Importantly, immunizing with a full-length Gag greatlyboosted the pre-existing CE responses. Hence, exposure to virus mightalso have the effect of boosting CE responses in CE-vaccinatedindividuals.

In a recent paper, Stephenson et al, [17] compared responses offull-length molecules to their conserved elements (Gag, Pol, and Env)vaccine and concluded that the conserved element vaccine did not provideany benefit (breadth or magnitude). In contrast, we demonstrated a clearbenefit from the CE vaccine, showing increased breadth and magnitude ofresponses. The difference between the studies may substantively by dueto our more strict definition and selection of CE, which, in contrast toothers, were selected in part by their association with virus control[34], further supporting their immunological relevance.

Previous analyses of HIV-1 infected persons with different HLAhaplotypes demonstrated the presence of CE-specific T cells during thechronic phase of infection [20,34]. Higher avidity CTL responses inthese regions were identified in HIV controllers and detailed analysisof the responses demonstrated that, for most epitopes analyzed,controllers were able to recognize more peptide variants [34]. Thisindicates that TCR promiscuity could be beneficial for the recognitionof epitopes with mismatched amino acids resulting in better control ofviral replication and prevention of escape mutants. These data alsosuggest that high avidity CE-specific responses are a potentialcorrelate of HIV control. It is not clear why vaccination withfull-length Gag generates poor CE responses (in mice or macaques), whilethese responses are detected in chronic HIV infection. It would be ofinterest to study different vaccination regimens and also to examine thetime of development of CE responses during natural infection. Thedifference in elicited immune response is reminiscent of a previousreport by Ferrari et al. [84], who showed that the immunodominantp17^(gag) SL9 response identified in HLA-A*0201 infected persons couldnot be induced upon ALVAC-gag vaccination in these haplotype-selectedvolunteers, although this epitope has been implicated in the Sieveeffect observed in the STEP HIV vaccine trial [85]. Both studies suggestthat there may be differences between vaccine-induced andinfection-induced cellular responses that should be taken intoconsideration for successful vaccine design; they also highlight thepotential immunodominant decoy effect of a full-length immunogen design.

Impaired immunogenicity of the conserved elements in the context of thenatural protein sequence could be due to the presence of variableregions, which may exert an immunodominant decoy effect preventing therecognition of the conserved epitopes. This possibility is supported bya recent study where a bias was found towards less-conserved regions inHIV-1 Ad5 gag/pol/nef vaccinated human volunteers [86]. Generation ofresponses mainly outside of the conserved elements by full-length Gagsuggested an immunodominant decoy effect. In this context, the successof our p24CE DNA prince-p55^(gag) DNA boost vaccine strategy is of greatimportance, because it showed strong boosting of pre-existingCE-specific cellular and humoral responses in macaques. Upon gag DNAboost, we report both a robust increase of the pre-existing CE-specificresponses as well as development of de novo responses to regions outsidethe CE. These data imply that the immunodominance exerted by Gagepitopes outside of CE was lost in the context of pre-existingCE-specific responses.

The impaired immunogenicity of the conserved elements when expressed inthe context of the complete Gag could in principle be related tosuboptimal processing and presentation of the CE peptides, preventingefficient priming of adaptive immune responses. However, our p24CEprime-Gag boost study clearly demonstrates that processing of thefull-length Gag protein produces a collection of CE-containing peptidesthat are recognized by T cells. We speculate that recognition ofMHC-peptide complexes is less stringent for boosting memory T cellclones than for priming nave T cells. Similar to the observations oncellular immunity, full-length Gag boosted pre-existing B cellresponses, while failing to prime the development of de novo antibodiesable to recognize the CE protein. These findings support the conceptthat proper processing of CE-containing peptides from the native Gagprotein takes place, and that these sets of CE containing peptides areable to potently augment pre-existing responses to different extents.Together, these findings point to a critical difference in T cellrecognition of these peptides where a clear distinction betweenantigen-experienced and nave T cells is noted.

The question then arises whether a T cell vaccine can benefit from theresponses elicited by selected T cell epitopes. A previous report [87]demonstrated the potency of T cell immunity in the absence of Env. Infact, a recent paper by Mudd et al. [88] showed that a T cell vaccinethat induced Mamu-B*08-restricted CD8⁺ T-cell responses targeting 3different viral epitopes elicited responses able to control SIV_(mac239)replication. Since our CE DNA vaccine was selected to highly restrictedsequences and haplotype-independent, it is plausible that they too couldinduce such potent responses, which will be addressed in future studies.

The presented results contribute significantly to the development ofimproved vaccine candidates against HIV targeting the immune responsesto essential highly conserved regions for the virus. We hypothesize thatcellular immune responses targeting conserved regions of HIV and otherhighly variable pathogens, which do not allow rapid escape mutationswithout significant loss of viral fitness, are more likely to beprotective [32-34]. Since there is evidence that vaccine-inducedresponses can change upon HIV infection resulting in virus escape inhumans [89], a selection of strictly conserved elements is of greatimportance for the design of an effective vaccine. Such a selectionshould also avoid epitopes that may act as immunodominant decoys. Thus,a successful vaccine should be able to generate potent cross-cladespecific humoral and cellular responses against conserved regions of thevirus. Our results provide an effective strategy to overcomerestrictions associated with immunodominance, while improving themagnitude and breadth of responses, especially those against conservedregions, minimizing the possibility of viral escape while increasing therecognition of naturally occurring divergent HIV strains. These resultsindicate that a vaccine candidate should be designed to extend thisconcept to the entire HIV proteome. Since the macaque model was ingeneral shown to provide a similar response hierarchy to that obtainedupon vaccination of humans comparing different vaccine platforms [90],our macaque study supports the evaluation of the novel CE vaccinestrategies in humans.

Materials and Methods

DATA Vectors

The p24CE plasmids pSP-p24CE1 (plasmid 234H) and pSP-p24CE2 (plasmid235H) have been described [57] and contain the human GM-CSF signalpeptide at the N-terminus of the expression-optimized p24CE open readingframe. Briefly, the 7 CE were collinearly assembled in the orderCE2-3-4-5-6-7-1 to avoid a strongly hydrophobic N-terminal CE1, and wereconnected via short linker sequences designed for efficient proteolyticcleavage [91,92]. The COT-M p55^(gag) [93] DNA (plasmid 222H) expressesthe full-length Gag from an RNA/codon optimized gene. The IL-12 DNA(plasmid AG157) produces the rhesus macaque IL-12 cytokine from anoptimized expression vector [94,95]. The vaccine vector CMVkan [81] iscomprised of a plasmid backbone optimized for growth in bacteria, thehuman cytomegalovirus (CMV) promoter without introns, the optimizedp24CE or gag genes, the bovine growth hormone (BGH) polyadenylationsite, and the kanamycin resistance gene. Endotoxin-free DNAs (Qiagen,Valencia, Calif.) were prepared according to the manufacturer'sprotocol.

Example 3 Additional Conserve Element Polypeptides

Alternative conserved elements were designed (FIG. 13). Briefly, CE1 wasextended to provide a CE8. CE2 was extended to provide CE9. In thisconstruct CE7 was removed. Accordingly, there are six conserve elementsin the CE polypeptide. There were two variants of the conserve elementpolypeptide where one amino acid is changed in CE8 and CE9. Two versionswere designed having different arrangements of the CEs. In the versiontermed “p24CEc” (p24CE1c and p24Ce2c), the order is CE8-9-2-3-4-6. Inthe version terms “p24CEd” (p24CE1d and p24CE2d), the order isCE9-3-4-5-6-8.

Example 4 Illustrative Data from 3 Different Vaccination Prime-BoostSchedules

We tested three different vaccination strategies in which the order ofconserved element vaccines and full-length gag vaccine was varied. Thethree protocols are shown in FIG. 20:

-   1. p24CE prime followed by p55gag boost-   2. p55gag prime followed by p24CE boost-   3. combination of p24CE and p55gag in all vaccinations.

The animals received 1 mg of each DNA. For p24CE, SP-p24CE1 and SPp24CE2were used. The DNA was administered via the intramuscular route followedby in vivo electroporation. FIG. 21 shows the cellular immune responsesbefore and after the boost. Cellular immune responses were measured withpeptides (15-mer overlapping by 11 amino acids) spanning the completep24gag. This analysis showed responses in all the vaccinated animals.

The animals were also analyzed for CE-specific responses using a peptidepool (mixture of 10-me peptide overlapping by 9 amino acids and 15-meroverlapping by 11 amino acids) spanning the 7 CE. All the p24CEvaccinated animals showed positive responses. In contrayst, Only 5 ofthe 11 gag DNA vaccinated animals showed responses (data shown). Threeof 4 animals with the combination vaccine (p24CE+p55^(gag)) showedpositive responses. After boosting by p55gag DNA, the p24CE primedanimals significantly increased CE-specific responses. p24CE DNA boostincreased the responses of the gag vaccinated animals (no significantincrease). A third vaccination of the animals vaccinated by thecombination (p24CE+p55^(gag)) did not consistently further increaseresponses.

FIG. 22 shows the analysis of the responses to individual CE. Theresponses to each CE were mapped in all the animals using CE-specificpeptides (mixture of 10-mer peptide overlapping by 9 amino acids and15-mer peptides overlapping by 11 amino acids) for each CE. The numberof CE that showed positive responses per animal are shown. The p24CEprimed animals had 100% response rates with a range of 1-3 CE/animal andmedian of 3 CE per animal. The gag DNA primed animals have a responserate of 45% with a range of 0-2 CE/animal. The animals that received thecombination vaccine are More similar to the CE primed animals.

FIG. 23 shows that different vaccine strategies induced similar levelsof p27gag antibody responses. Binding antibody titers were measured inthe plasma of macaques by ELISA.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

All publications, accession numbers, patents, and patent applicationscited in this specification are herein incorporated by reference as ifeach was specifically and individually indicated to be incorporated byreference.

REFERENCES

-   1. Nickle D C, Rolland M, Jensen M A, Pond S L, Deng W, et    al. (2007) Coping with viral diversity in HIV vaccine design. PLoS    Comput Biol 3: e75.-   2. Nickle D C, Jojic Heckerman D, Jojic V, Kirovski D, et al. (2008)    Comparison of immunogen designs that optimize peptide coverage:    reply to Fischer et al. PLoS Comput Biol 4: e25.-   3. Barouch D H, O'Brien K L, Simmons N L, King S L, Abbink P, et al.    (20)0) Mosaic HIV-1 vaccines expand the breadth and depth of    cellular immune responses in rhesus monkeys. Not Med 16: 319-323.-   4. Santra S, Liao H X, Zhang R, Muldoon M, Watson S, et al. (2010)    Mosaic vaccines elicit CD8+ T lymphocyte responses that confer    enhanced immune coverage of diverse HIV strains in monkeys. Nat Med    16: 324-328.-   5. Fischer W, Perkins S, Theiler J, Bhattacharya T, Yusim K, et    al. (2007) Polyvalent vaccines for optimal coverage of potential    T-cell epitopes in global HIV-1 variants. Nat Med 13: 100-106.-   6. Fischer W, Liao H X, Haynes B F, Letvin N L, Korber B (2008)    Coping with viral diversity in HIV vaccine design: a response to    Nickle et al. PLoS Comput Biol. 4: e15; author reply e25,-   7. Doric-Rose N A, Learn G H, Rodrigo A G. Nickle D C, Li F. et al.    (2.005) Human immunodeficiency virus type 1 subtype B ancestral    envelope protein is functional and elicits neutralizing antibodies    in rabbits similar to those elicited by a circulating subtype B    envelope. J Virol 79: 11214-11224.-   8. Mullins J I, Nickle D C, Heath L, Rodrigo A G, Learn G H (2004)    immunogen sequence: the fourth tier of AIDS vaccine design. Expert    Rev Vaccines 3: S151-159.-   9. Nickle D C, Jensen M A, Gottlieb G S, Shriner D, Learn G H, et    al. (2003) Consensus and ancestral state HIV vaccines. Science 299:    1515-1518; author reply 1515-1518.-   10. Dahirel V, Shekhar K, Pereyra F, Miura T, Artyomov M, et    al. (2011) Coordinate linkage of HIV evolution reveals regions of    immunological vulnerability. Proc Natl Acad Sci USA 108:    11530-11535.-   11. Letourneau S, Im E J, Mashishi T, Brereton C, Bridgeman A, et    al. (2007) Design and pre-clinical evaluation of a universal HIV-1    vaccine. PLoS One 2: e984.-   12. Rosario M, Bridgeman A, Quakkelaar E D, Quigley M E, Hill B J,    et al. (2010) Long peptides induce polyfunctional T cells against    conserved regions of HIV-1 with superior breadth to single-gene    vaccines in macaques. Eur J Immunol 40: 1973-1984.-   13. De Groot A S, Rivera D S, McMurry J A, Buus S, Martin W (2008)    Identification of immunogenic HLA-B7 “Achilles' heel” epitopes    within highly conserved regions of HIV. Vaccine 26: 3059-3071.-   14. Wilson C C, McKinney D, Anders M, MaWhinney S, Forster J, et    al. (2003) Development of a DNA vaccine designed to induce cytotoxic    T lymphocyte responses to multiple conserved epitopes in HIV-1. J    Immunol 171: 5611-5623.-   15. Kaufman D R, Li F, Cruz A N, Self S G, Barouch D H (2012) Focus    and breadth of cellular tune responses elicited by a heterologous    insert prime-boost vaccine regimen in rhesus monkeys. Vaccine 30:    506-509.-   16. Almeida R R, Rosa D S, Ribeiro S P, Santana V C, Kallas E G, et    al. (2012) Broad and cross-glade CD4+ T-cell responses elicited by a    DNA vaccine encoding highly conserved and promiscuous HIV-1 M-group    consensus peptides. PloS One 7: e45267.-   17. Stephenson K E, SanMiguel A, Simmons N L, Smith K, Lewis M G, et    al. (2012) Full-length HIV-1 immunogens induce greater magnitude and    comparable breadth of T lymphocyte responses to conserved HIV-1    regions compared with conserved-region-only HIV-1 immunogens in    rhesus monkeys. J Virol 86: 11434-11440.-   18. Lichterfeld. M, Yu X G, Le Gall S, Altfeld M (2005)    Immunodominance of HIV-1-specific CD8(+) T-cell responses in acute    HIV-1 infection: at the crossroads of viral and host genetics.    Trends Immunol 26: 166-171.-   19. Friedrich T C, Valentine L E, Yant L J, Rakasz E G, Piaskowski S    M, et al. (2007) Subdominant CD8+ T-cell responses are involved in    durable control of AIDS virus replication. J Virol 81: 3465-3476.-   20. Liu Y, McNevin J, Rolland M, Zhao H, Deng W, et al. (2009)    Conserved HIV-1 epitopes continuously elicit subdominant cytotoxic    T-lymphocyte responses. J infect Dis 200: 1825-1833,-   21. Liu J, Ewald B A, Lynch D M, Nanda A, Sumida S M, et al. (2006)    Modulation of DNA vaccine-elicited CD8+ T-lymphocyte, epitope    immunodominance hierarchies. J Virol 80: 11991-11997.-   22. Frahm. N, Kiepiela P, Adams S, Linde C H, Hewitt H S, et    al. (2006) Control of human immunodeficiency virus replication by    cytotoxic T lymphocytes targeting subdominant epitopes. Nat Immunol    7: 173-178.-   23. Bockl K, Wild J, Bredl S, Kindsmuller K, Kostler J, et    al. (2012) Altering an Artificial Gagpolnef Polyprotein and Mode of    ENV Co-Administration Affects the Immunogenicity of a Clade C HIV    DNA Vaccine. PLoS One 7: e34723.-   24. Iversen A K, Stewart-Jones G, Learn G H, Christie N,    Sylvester-Hviid C, et al. (2006) Conflicting selective forces affect    T cell receptor contacts in an immunodominant human immunodeficiency    virus epitope. Nat Immunol 7: 179-189.-   25. Schneidewind A, Brumme Z L, Brumme C J, Power K A, Reyor L L, et    al. (2009) Transmission and long-term stability of compensated CD8    escape mutations. J Virol 83: 3993-3997.-   26. Altfeld M, Kalife E T, Qi Y, Streeck H, Lichterfeld M, et    al. (2006) HLA Alleles S Associated with Delayed Progression to AIDS    Contribute Strongly to the Initial CD8(+) T Cell Response against    HIV-1, PLoS Med 3: e403,-   27. Friedrich D, Jalbert E, Dinges W L, Sidney J, Sette A, et    al. (2011) Vaccine-induced HIV-specific CD8+ T cells utilize    preferential HLA alleles and target-specific regions of HIV-1. J    Acquir Immune Defic Syndr 58: 248-252.-   28. Maurer K, Harrer E G, Goldwich A, Eismann K, Bergmann 5, et    al. (2008) Role of cytotoxic T-lymphocyte-mediated immune selection    in a dominant human leukocyte antigen-B8-restricted cytotoxic    T-lymphocyte epitope in Nef. J Acquir Immune Defic Synth 48:    133-141.-   29. Toapanta F R, Craigo J K, Montelaro R C, Ross T M (2007)    Reduction of anti-HIV-1 Gag immune responses during co-immunization:    immune interference by the HIV-1 envelope. Current HIV research 5:    199-209.-   30. Morozov V A, Morozov A V, Semaan M, Denner J (2012) Single    mutations in the transmembrane envelope protein abrogate the    immunosuppressive property of HIV-1. Retrovirology 9: 67,-   31. Hovav A H, Santosuosso M, Bivas-Benita M, Plair A, Cheng A, et    al. (2009) X4 human immunodeficiency type 1 gp120 down-modulates    expression and immunogenicity of codelivered antigens. Journal of    virology 83: 10941-10950.-   32. Rolland M, Nickle D C, Mullins J I (2007) HIV-1 group M    conserved elements vaccine. PLoS Pathog 3: e157.-   33. Niu L, Termini J M, Kanagavelu S K, Gupta. S, Rolland M M, et    al. (2011) Preclinical evaluation of HIV-1 therapeutic ex vivo    dendritic cell vaccines expressing consensus Gag antigens and    conserved Gag epitopes. Vaccine 29: 2110-2119.-   34. Mothe B, Llano A, Ibarrondo J, Zamarreno J, Schiaulini M, et    al. (2012) CTL responses of high functional avidity and broad    variant cross-reactivity are associated with HIV control. PLoS One    7: e29717.-   35. Herbeck J T, Nickle D C, Learn G H, Gottlieb G S, Curlin M E, et    al, (2006) Human immunodeficiency virus type 1 env evolves toward    ancestral states upon transmission to a new host. J Virol 80:    1637-1644.-   36. Duda A, Lee-Turner L, Fox J, Robinson N, Dustan S, et al. (2009)    HLA-associated clinical progression correlates with epitope    reversion rates in early human immunodeficiency virus infection. J    Virol 83: 1228-1239,-   37. Kent S J, Fernandez C S, Dale C J, Davenport M P (2005)    Reversion of immune escape HIV variants upon transmission: insights    into effective viral immunity. Trends Microbiol 13: 243-246.-   38. Li B, Gladden A D, Altfeld M, Kaldor J M, Cooper D A, et    al. (2007) Rapid reversion of sequence polymorphisms dominates early    human immunodeficiency virus type 1 evolution. J Virol 81: 193-201.-   39. Martinez-Picado J, Prado J G, Fry E E, Pfafferott K, Leslie A,    et al. (2006) Fitness cost of escape mutations in p24 Gag in    association with control of human immunodeficiency virus type 1. J    Virol 80: 3617-3623.-   40. Peyerl F W, Bazick H S, Newberg M H, Barouch D H, Sodroski J, et    al. (2004) Fitness costs limit viral escape from cytotoxic T    lymphocytes at a structurally constrained epitope. J Virol 78:    13901-13910.-   41. Liu Y, McNevin J, Zhao H, Tebit D M, Troyer R M, et al. (2007)    Evolution of human immunodeficiency virus type 1 cytotoxic    T-lymphocyte epitopes: fitness-balanced escape. J Virol 81:    12179-12188.-   42. Troyer R M, Collins K R, Abraha A, Fraundorf E, Moore D M, et    al. (2005) Changes in human immunodeficiency virus type 1 fitness    and genetic diversity during disease progression. J Virol 79:    9006-9018,-   43. Kiepiela P, Ngumbela K, Thohakgaie C, Ramduth D, Honeyborne I,    et al. (2007) CD8+ T-cell responses to different HIV proteins have    discordant associations with viral load. Nat Med 13: 46-53.-   44. Honeyborne I, Prendergast A, Pereyra F, Leslie A, Crawford H, et    al. (2007) Control of human immunodeficiency virus type 1 is    associated with HLA-B*13 and targeting of multiple gag-specific CD8+    epitopes. J Virol 81: 3667-3672.-   45. Schneidewind A, Brockman M A, Yang R, Adam R I, Li B, et    al. (2007) Escape from the dominant HLA-B27-restricted cytotoxic    T-lymphocyte response in Gag is associated with a dramatic reduction    in human immunodeficiency virus type 1 replication. J Virol 81:    12382-12393.-   46. Ngumbela K C, Day C L, Mncube Z, Nair K. Ramduth D, et    al. (2008) Targeting of a CD8+ T cell env epitope presented by    HLA-B*5802 is associated with markers of HIV disease progression and    lack of selection pressure. AIDS Res Hum Retroviruses 24: 72-82.-   47. Rolland M, Heckerman D, Deng W, Rousseau C M, Coovadia H, et    al. (2008) Broad and Gag-biased HIV-1 epitope repertoires are    associated with lower viral loads. PLoS One 3: e1424.-   48. Masemola A, Mashishi T, Khoury G, Mohube P, Mokgotho P, et    al. (2004) Hierarchical targeting of subtype C human    immunodeficiency virus type 1 proteins by CD8+ T cells: correlation    with viral load. J Virol 78: 3233-3243.-   49. Mothe B, Ibarrondo J, Llano A, Brander C (2009) Virological,    immune and host genetics markers in the control of HIV infection.    Dis Markers 27: 105-120.-   50. Zuniga R, Lucchetti A, Galvan P, Sanchez S, Sanchez C, et    al. (2006) Relative dominance of Gag p24-specific cytotoxic T    lymphocytes is associated with human immunodeficiency virus control.    J Virol 80: 3122-3125.-   51. Mothe B, Llano A, Ibarrondo J, Daniels M, Miranda C, et    al. (2011) Definition of the viral targets of protective    HIV-1-specific T cell responses. J Transl Med 9: 208.-   52. Assarsson E, Sidney J, Oseoff C, Pasquetto V, Bui H H, et    al. (2007) A quantitative analysis of the variables affecting the    repertoire of T cell specificities recognized after vaccinia virus    infection. J Immunol 178: 7890-7901.-   53. Altfeld M, Allen T M (2006) Hitting HIV where it hurts: an    alternative approach to HIV vaccine design. Trends Immunol 27:    504-510.-   54. Rosario M, Borthwick N, Stewart-Jones G B, Mbewe-Mvula A,    Bridgeman A, et al. (2012) Prime-boost regimens with adjuvanted    synthetic long peptides elicit T cells and antibodies to conserved    regions of HIV-1 in macaques. AIDS 26: 275-284.-   55. Ribeiro S P, Rosa. D S, Fonseca S G, Mairena. E C, Postol E, et    al. (2010) A vaccine encoding conserved promiscuous HIV CD4 epitopes    induces broad T cell responses in nice transgenic to multiple common    HLA class II molecules. PloS One 5: e11072.-   56. Rosa D S, Ribeiro S P, Almeida R R, Mairena E C, Postol E, et    al. (2011) A DNA vaccine encoding multiple HIV CD4 epitopes elicits    vigorous polyfunctional, long-lived CD4+ and CD8+ T cell responses.    PloS One 6: e16921.-   57. Kulkarni V, Rosati M, Valentin A, Ganneru B, Singh A K, et al.    An HIV-1 p24gag derived conserved element DNA vaccine increased the    breadth of immune response in mice. submitted.-   58. Winstone N, Wilson A J, Morrow G, Boggiano C, Chiuchiolo M J, et    al. (2011) Enhanced control of pathogenic SIV mac239 replication in    macaques immunized with a plasmid IL12 and a DNA prime, viral vector    boost vaccine regimen. J Virol 85: 9578-9587,-   59. Aihara H, Miyazaki J (1998) Gene transfer into muscle by    electroporation in vivo. Nat Biotechnol 16: 867-870.-   60. Mathiesen I (1999) Electropermeabilization of skeletal muscle    enhances gene transfer in vivo. Gene Ther 6: 508-514.-   61. Prud'homme G J, Y, Khan A S, Draghia-Akli R (2006)    Electroporation-enhanced nonviral gene transfer for the prevention    or treatment of immunological, endocrine and neoplastic diseases.    Curr Gene Ther 6: 243-273,-   62. Rizzuto G, Cappelletti M, Maione D, Savino R, Lazzaro D, et    al. (1999) Efficient and regulated erythropoietin production by    naked DNA injection and muscle electroporation. Proc Natl Acad Sci    USA 96: 6417-6422.-   63. Wang Z, Troilo P J, Wang X, Griffiths T G, Pacchione S J, et    al. (2004) Detection of integration of plasmid DNA into host genomic    DNA following intramuscular injection and electroporation. Gene Ther    11: 711-721.-   64. Widera G, Austin M, Rabussay D, Goldbeck C, Barnett S W, et    al. (2000) increased DNA vaccine delivery and immunogenicity by    electroporation in vivo. J Immunol 164: 4635-4640.-   65. Often G, Schaefer M, Doe B, Liu H, Srivastava I, et al. (2004)    Enhancement of DNA vaccine potency in rhesus macaques by    electroporation. Vaccine 22: 2489-2493.-   66. Often G R, Schaefer M, Doe B, Liu H, Megede J Z, et al. (2006)    Potent immunogenicity of an HIV-1 gag-pol fusion DNA vaccine    delivered by in vivo electroporation. Vaccine 24: 4503-4509.-   67. Rosati M, Valentin A, Jalah R, Patel V, von Gegerfelt A, et    al, (2008) Increased immune responses in rhesus macaques by DNA    vaccination combined with electroporation. Vaccine 26:5223-5229.-   68. Luckay A, Sidhu M K, Kjeken R, Megati S, Chong S Y, et    al. (2007) Effect of plasmid DNA vaccine design and in vivo    electroporation on the resulting vaccine-specific immune responses    in rhesus macaques. J Virol 81: 5257-5269.-   69. Hirao L A, Wu L, Khan A S, Hokey D A, Yan J, et al, (2008)    Combined effects of IL-12 and electroporation enhances the potency    of DNA vaccination in macaques. Vaccine 26: 3112-3120.-   70. Rosati M, Bergamaschi C, Valentin A, Kulkarni V, Jalah R, et    al. (2009) DNA vaccination in rhesus macaques induces potent immune    responses and decreases acute and chronic viremia after SIVmac251    challenge. Proc Natl Acad Sci USA 06: 1:5831-15836.-   71. Patel V, Valentin A, Kulkarni V, Rosati M, Bergamaschi C, et    al. (2010) Long-lasting humoral and cellular immune responses and    mucosal dissemination after intramuscular DNA immunization. Vaccine    28: 4827-4836.-   72. Vasan S, Schlesinger S J, Chen Z, Hurley A, Lombardo A, et    al. (2010) Phase 1 safety and immunogenicity evaluation of ADMVA, a    multigenic, modified vaccinia Ankara-HIV-1 B′/C candidate vaccine.    PLoS One 5: e8816.-   73. Nasioulas G, Zolotukhin A S, Tabernero C, Solomin L, Cunningham    C P, et al. (1994) Elements distinct from human immunodeficiency    virus type 1 splice sites are responsible for the Rev dependence of    env mRNA. J Virol 68: 2986-2993.-   74. Schneider R, Campbell M, Nasioulas G, Felber B K, Pavlakis G    N (1997) Inactivation of the human immunodeficiency virus type 1    inhibitory elements allows Rev-independent expression of Gag and    Gag/protease and particle formation. J Virol 71: 4892-4903.-   75. Schwartz S, Campbell M, Nasioulas G, Harrison J, Felber B K, et    al, (1992) Mutational inactivation of an inhibitory sequence in    human immunodeficiency virus type 1 results in Rev-independent gag    expression. J Virol 66: 7176-7182.-   76. Schwartz S, Felber B K, Pavlakis G N (1992) Distinct RNA    sequences in the gag region of human immunodeficiency virus type 1    decrease RNA stability and inhibit expression in the absence of Rev    protein. J Virol 66: 150-159.-   77. Andre S, Seed B, Eberle J. Schraut W, Bultmann A, et al. (1998)    Increased immune response elicited by DNA vaccination with a    synthetic gp120 sequence with optimized codon usage. J Virol 72:    1497-1503.-   78. Wagner R, Graf M, Bieler K, Wolf H, Grunwald T, et al. (2000)    Rev-independent expression of synthetic gag-pol genes of human    immunodeficiency virus type 1 and simian immunodeficiency virus:    implications for the safety of lentiviral vectors. Hum Gene Ther 11:    2403-2413.-   79. Graf M, Deml L, Wagner R (2004) Codon-optimized genes that    enable increased heterologous expression in mammalian cells and    elicit efficient immune responses in mice after vaccination of naked    DNA. Methods Mol Med 94: 197-210.-   80. Kulkarni V, Jalah R, Ganneru B, Bergamaschi C, Alicea C, et    al. (2011) Comparison of immune responses generated by optimized DNA    vaccination against SIV antigens in mice and macaques. Vaccine 29:    6742-6754.-   81. Rosati M, von Gegerfelt A, Roth P, Alicea C, Valentin A, et    al. (2005) DNA vaccines expressing different forms of simian    immunodeficiency virus antigens decrease viremia upon SIVmac251    challenge. J Virol 79: 8480-8492.-   82. Valentin A, Chikhlikar P, Patel V, Rosati M, Maciel M, et    al. (2009) Comparison of DNA vaccines producing HIV-1 Gag and    LAMP/Gag chimera in rhesus macaques reveals antigen-specific T-cell    responses with distinct phenotypes. Vaccine 27: 4840-4849.-   83. Vojnov L, Bean A T, Peterson E J, Chiuchiolo M J, Sacha J B, et    al. (201)) DNA/Ad5 vaccination with SIV epitopes induced    epitope-specific CD4 T cells, but few subdominant epitope-specific    CD8 T cells. Vaccine 29: 7483-7490.-   84. Ferrari G. Neal W, Ottinger J, Jones A M, Edwards B H, et    al. (2004) Absence of immunodominant anti-Gag p17 (SL9) responses    among Gag CTL-positive, HIV-uninfected vaccine recipients expressing    the HLA-A*0201 allele. J Immunol 173: 2126-2133.-   85. Rolland M, Tovanabutra S, deCamp A C, Frahm N, Gilbert P B, et    al. (2011) Genetic impact of vaccination on breakthrough HIV-1    sequences from the STEP trial. Nat Med 17: 366-171.-   86. Li F, Finnefrock A C, Dubey S A, Korber B T, Szinger J, et al.    Mapping HIV-1 vaccine induced T-cell responses: bias towards    less-conserved regions and potential impact on vaccine efficacy in    the Step study. PLoS One 6: e20479.-   87. Wilson N A, Keele B F, Reed J S, Piaskowski S M, MacNair C E, et    al, (2009) Vaccine-induced cellular responses control simian    immunodeficiency virus replication after heterologous challenge. J    Virol 83: 6508-6521.-   88. Mudd P A, Martins M A, Ericsen A J, Tully D C, Power K A, et    al. (2012) Vaccine-induced CD8+ T cells control AIDS virus    replication. Nature 491: 129-133.-   89. Betts M R, Exley B, Price D A, Bansal A, Camacho Z T, et    al. (2005) Characterization of functional and phenotypic changes in    anti-Gag vaccine-induced T cell responses and their role in    protection after HIV-1 infection, Proc Natl Acad Sci U.S.A. 102:    4512-4517.-   90. Bett A J, Dubey S A, Mehrotra D V, Guan L, Long R, et al. (2010)    Comparison of T cell immune responses induced by vectored HIV    vaccines in non-human primates and humans. Vaccine 28: 7881-7889,-   91. Le Gall S, Stamegna P, Walker B D (2007) Portable flanking    sequences modulate CTL epitope processing. J Clin Invest 117:    3563-3575.-   92. Zhang S C, Martin E, Shimada M, Godfrey S B, Fricke J, et    al. (2012) Aminopeptidase Substrate Preference Affects HIV Epitope    Presentation and Predicts Immune Escape Patterns in HIV-Infected    Individuals, J Immunol 188: 5924-5934.-   93. Rolland M, Jensen M A, Nickle D C, Van J, Learn G H, et    al. (2007) Reconstruction and function of ancestral center-of-tree    human immunodeficiency virus type 1 proteins. J Virol 81: 8507-8514.-   94. Jalah R, Patel V, Kulkarni V, Rosati M, Alicea C, et al. (2012)    IL-12 DNA as molecular vaccine adjuvant increases the cytotoxic T    cell responses and breadth of humoral immune responses in SIV DNA    vaccinated macaques. Hum Vaccin Immunother 8: 1620-1629.-   95. Jalah R, Rosati M, Ganneru B, Pilkington G R, Valentin A, et    al. (2013) The p40 Subunit of IL-12 Promotes Stabilization and    Export of the p35 Subunit: Implications for Improved IL-12 Cytokine    Production. J Biol Chem in press.-   96. Ranke T, McMichael A (1999) Pre-clinical development of a    multi-CTL epitope-based DNA prime MVA boost vaccine for AIDS.    Immunol Lett 66: 177-181,-   97. Pornillos O, Ganser-Pornillos B K, Kelly B N, Hua Y, Whitby F C,    et al. (2009) X-ray structures of the hexameric building block of    the HIV capsid. Cell 137: 1282-1292.-   98. Chikhlikar P, de Arruda L B, Maciel M, Silvera P, Lewis M G, et    al. (2006) DNA encoding an HIV-1 Gag/human lysosome-associated    membrane protein-1 chimera elicits a broad cellular and Immoral    immune response in Rhesus macaques. PLoS ONE 1: e135.-   99. de Arruda L B, Chikhlikar P R, August J T, Marques E T (2004)    DNA vaccine encoding human immunodeficiency virus-1 Gag, targeted to    the major histocompatibility complex II compartment by    lysosomal-associated membrane protein, elicits enhanced long-term    memory response. Immunology 112: 126-133.-   100. Marques E T, Jr., Chikhlikar P, de Arruda L B, Leao I C, Lu Y,    et al. (2003) HIV-1 p55Gag encoded in the lysosome-associated    membrane protein-1 as a DNA plasmid vaccine chimera is highly    expressed, traffics to the major histocompatibility class II    compartment, and elicits enhanced immune responses. J Biol Chem 278:    37926-37936.-   101. Valentin A, Chikhlikar P, Patel V, Rosati M, Maciel M, et    al. (2009) Comparison of DNA vaccines producing HIV-1 Gag and    LAMP/Gag chimera in rhesus macaques reveals antigen-specific T-cell    responses with distinct phenotypes. Vaccine 27: 4840-4849.-   102. Qiu J T, Song R, Dettenhofer M, Tian C, August T, et al. (1999)    Evaluation of novel human immunodeficiency virus type 1 Gag DNA    vaccines for protein expression in mammalian cells and induction of    immune responses. J Virol 73: 9145-9152.-   103. von Gege felt A, Valentin A, Alicea C, Van Rompay K K, Marthas    M L, et al. (2010) Emergence of simian immunodeficiency    virus-specific cytotoxic CD4+ T cells and increased humoral    responses correlate with control of rebounding viremia in    CD8-depleted macaques infected with Rev-independent live-attenuated    simian immunodeficiency virus. J Immunol 185: 3348-3358.-   104. Lazard E, Kadie C, Stamegna P, Zhang S C, Gourdain P, et    al. (2011) Variable HIV peptide stability in human cytosol is    critical to epitope presentation and immune escape. J Clin Invest    121: 2480-2492.-   105. Schwartz S, Felber B K, Pavlakis G N (1992) Mechanism of    translation of monocistronic and multicistronic human    immunodeficiency virus type 1 mRNAs. Mol Cell Biol 12: 207-219.-   106. Rolland M, Jensen M A, Nickle D C, Yan J, Learn G H, et    al. (2007) Reconstruction and function of ancestral center-of-tree    human immunodeficiency virus type 1 proteins. J Virol 81: 8507-8514.

Table of Illustrative Conserved Element Sequencesp24 Gag conserved elements for p24CE1 vaccine (“also referred to as Core1”):conserved element 1 (CE1) SEQ ID NO: 1 ISPRTLNAWVKVconserved element 2 (CE2) SEQ ID NO: 2 VIPMFSALSEGATPQDLNconserved element 3 (CE3) SEQ ID NO: 3 VGGHQAAMQMLKDTINEEAAEWDRconserved element 4 (CE4) SEQ ID NO: 4 PRGSDIAGTTSTLQEQIGWconserved element 5 (CE5) SEQ ID NO: 5 KRWIILGLNKIVRMYSPTSIconserved element 6 (CE6) SEQ ID NO: 6 YVDRFYKTLRAEQAconserved element 7 (CE7) SEQ ID NO: 7 LEEMMTACQGVGGPGHKp24 Gag conserved elements for p24CE2 vaccine (“also referred to as Core2”):conserved element 1 (CE1) SEQ ID NO: 8 LSPRTLNAWVKVconserved element 2 (CE2) SEQ ID NO: 9 VIPMFTALSEGATPQDLNconserved element 3 (CE3) SEQ ID NO: 10 VGGHQAAMQMLKETINEEAAEWDRconserved element 4 (CE4) SEQ ID NO: 11 PRGSDIAGTTSTLQEQIAWconserved element 5 (CE5) SEQ ID NO: 12 KRWIILGLNKIVRMYSPVSIconserved element 6 (CE6) SEQ ID NO: 13 YVDRFFKTLRAEQAconserved element 7 (CE7) SEQ ID NO: 14 LEEMMTACQGVGGPSHKp24 Gag conserved elements for p24CE1 vaccine (“also referred to as Core1”):SEQ ID NO: 15VIPMFSALSEGATPQDLNAAVGGHQAAMQMLKDTINEEAAEWDRAAAEPRGSDIAGTTSTLQEQIGWAAAKRWIILGLNKIVRMYSPTSIAAKYVDRFYKTLRAEQAAGLEEMMTACQGVGGPGHKAAISPRTLNAWVKVp24 Gag conserved elements for p24CE2 vaccine (“also referred to as Core2”):SEQ ID NO: 16VIPMFTALSEGATPQDLNAAVGGHQAAMQMLKETINEEAAEWDRAAAEPRGSDIAGTTSTLQEQIAWAAAKRWIILGLNKIVRMYSPVSIAAKYVDRFFKTLRAEQAAGLEEMMTACQGVGGPSHKAALSPRTLNAWVKVNucleic acid construct encoding Core1 plus Core2 (p24CE1 +p24CE2) (306H) (genes underlined) SEQ ID NO: 17CCTGGCCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCATGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGATGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGGcgcgcgtcgacaagaaATGTGGCTCCAGAGCCTGCTACTCCTGGGGACGGTGGCCTGCAGCATCTCGGTCATCCCGATGTTCTCGGCGCTCAGCGAGGGAGCGACGCCGCAGGACCTGAACGCGGCCGTCGGAGGTCACCAGGCAGCGATGCAGATGCTGAAGGACACGATCAACGAGGAGGCGGCCGAGTGGGACCGGGCGGCAGCCGAGCCACGCGGTTCCGACATCGCGGGCACCACCTCGACGCTCCAGGAGCAGATCGGGTGGGCCGCAGCTAAGCGCTGGATCATCCTCGGGCTGAACAAGATCGTCCGGATGTACAGCCCGACGTCGATCGCTGCTAAGTACGTTGACCGGTTCTACAAGACCCTGAGGGCCGAGCAGGCGGCCGGACTGGAGGAGATGATGACCGCGTGCCAGGGGGTCGGTGGACCAGGGCACAAGGCCGCGATCTCGCCGCGCACGCTGAACGCGTGGGTGAAGGTCTGATAAgaattcgctagcggcgcgccagatctgatatcggatctGCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGGTACCCAGGTGCTGAAGAATTGACCCGGTTCCTCCTGGGCCAGAAAGAAGCAGGCACATCCCCTTCTCTGTGACACACCCTGTCCACGCCCCTGGTTCTTAGTTCCAGCCCCACTCATAGGACACTCATAGCTCAGGAGGGCTCCGCCTTCAATCCCACCCGCTAAAGTACTTGGAGCGGTCTCTCCCTCCCTCATCAGCCCACCAAACCAAACCTAGCCTCCAAGAGTGGGAAGAAATTAAAGCAAGATAGGCTATTAAGTGCAGAGGGAGAGAAAATGCCTCCAACATGTGAGGAAGTAATGAGAGAAATCATAGAATTTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCGGGGGGGGGGGGCGCTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATCATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTCAGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGATCATCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGATTATGATCgtcgaggatccggcgTTATCAGACCTTCACCCAGGCGTTGAGGGTGCGAGGCGAGAGGGCCGCCTTGTGCGACGGTCCTCCGACTCCCTGGCAGGCTGTCATCATCTCCTCGAGACCCGCGGCCTGCTCTGCCCTCAGCGTCTTGAAGAAGCGGTCTACGTATTTGGCCGCGATGCTGACTGGGCTGTACATCCTGACGATCTTGTTGAGGCCCAGGATGATCCAGCGCTTGGCTGCAGCCCAGGCGATCTGCTCCTGGAGGGTGCTGGTCGTGCCTGCGATGTCGCTACCCCTTGGCTCAGCTGCTGCCCTGTCCCACTCGGCTGCCTCCTCGTTGATGGTCTCCTTGAGCATCTGCATTGCCGCCTGGTGTCCACCGACCGCGGCGTTGAGGTCCTGCGGTGTCGCACCCTCACTGAGTGCGGTGAACATGGGGATGACCGAGATCGAGCACGCCACGGTCCCGAGTAGCAGGAGCGACTGCAGCCACATttcttccgtttaaacgtcgacagatccaaacGCTCCTCCGACGTCCCCAGGCAGAATGGCGGTTCCCTAAACGAGCATTGCTTATATAGACCTCCCATTAGGCACGCCTACCGCCCATTTACGTCAATGGAACGCCCATTTGCGTCATTGCCCCTCCCCATTGACGTCAATGGGGATGTACTTGGCAGCCATCGCGGGCCATTTACCGCCATTGACGTCAATGGGAGTACTGCCAATGTACCCTGGCGTACTTCCAATAGTAATGTACTTGCCAAGTTACTATTAATAGATATTGATGTACTGCCAAGTGGGCCATTTACCGTCATTGACGTCAATAGGGGGCGTGAGAACGGATATGAATGGGCAATGAGCCATCCCATTGACGTCAATGGTGGGTGGTCCTATTGACGTCAATGGGCATTGAGCCAGGCGGGCCATTTACCGTAATTGACGTCAATGGGGGAGGCGCCATATACGTCAATAGGACCGCCCATATGACGTCAATAGGTAAGACCATGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGATTGGCTATTGGCATTATGCCp24CE1 encoded by SEQ ID NO 17 (includes a GM-CSF signal peptide)SEQ ID NO: 18MWLQSLLLLGTVACSISVIPMFSALSEGATPQDLNAAVGGHQAAMQMLKDTINEEAAEWDRAAAEPRGSDIAGTTSTLQEQIGWAAAKRWIILGLNKIVRMYSPTSIAAKYVDRFYKTLRAEQAAGLEEMMTACQGVGGPGHKAAISPRTLNAWVKV p24CE2 encoded by SEQ ID NO 17 (includes a GM-CSF signal peptide)SEQ ID NO: 19MWLQSLLLLGTVACSISVIPMFTALSEGATPQDLNAAVGGHQAAMQMLKETINEEAAEWDRAAAEPRGSDIAGTTSTLQEQIAWAAAKRWIILGLNKIVRMYSPVSIAAKYVDRFFKTLRAEQAAGLEEMMTACQGVGGPSHKAALSPRTLNAWVKV LAMP-p24CE2 (202H) (gene underlined) SEQ ID NO: 20CCTGGCCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCATGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGATGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGcgggcgcgcgtcgactagcATGGCGCCCCGCAGCGCCCGGCGACCCCTGCTGCTGCTACTGCTGTTGCTGCTGCTCGGCCTCATGCATTGTGCGTCAGCAGCAATGTTTATGGTGAAAAATGGCAACGGGACCGCGTGCATAATGGCCAACTTCTCTGCTGCCTTCTCAGTGAACTACGACACCAAGAGTGGCCCTAAGAACATGACCCTTGACCTGCCATCAGATGCCACAGTGGTGCTCAACCGCAGCTCCTGTGGAAAAGAGAACACTTCTGACCCCAGTCTCGTGATTGCTTTTGGAAGAGGACATACACTCACTCTCAATTTCACGAGAAATGCAACACGTTACAGCGTTCAGCTCATGAGTTTTGTTTATAACTTGTCAGACACACCTTTTCCCCAATGCGAGCTCCAAAGAAATCAAGACTGTGGAATCTATAACTGACATCAGGGCAGATATAGATAAAAAATACAGATGTGTTAGTGGCACCCAGGTCCACATGAACAACGTGACCGTAACGCTCCATGATGCCACCATCCAGGCGTACCTTTCCAACAGCAGCTTCAGCAGGGGAGAGACACGCTGTGAACAAGACAGGCCTTCCCCAACCACAGCGCCCCCTGCGCCACCCAGCCCCTCGCCCTCACCCGTGCCCAAGAGCCCCTCTGTGGACAAGTACAACGTGAGCGGCACCAACGGGACCTGCCTGCTGGCCAGCATGGGGCTGCAGCTGAACCTCACCTATGAGAGGAAGGACAACACGACGGTGACAAGGCTTCTCAACATCAACCCCAACAAGACCTCGGCCAGCGGGAGCTGCGGCGCCCACCTGGTGACTCTGGAGCTGCACAGCGAGGGCACCACCGTCCTGCTCTTCCAGTTCGGGATGAATGCAAGTTCTAGCCGGTTTTTCCTACAAGGAATCCAGTTGAATACAATTCTTCCTGACGCCAGAGACCCGTGCCTTTAAAGCTGCCAACGGCTCCCTGCGAGCGCTGCAGGCCACAGTCGGCAATTCCTACAAGTGCAACGCGGAGGAGCACGTCCGTGTCACGAAGGCGTTTTCAGTCAATATATTCAAAGTGTGGGTCCAGGCTTTCAAGGTGGAAGGTGGCCAGTTTGGCTCTGTGGAGGAGTGTCTGCTGGACGAGAACAGCCTCGAGGATATCGTCATCCCGATGTTCACGGCGCTCAGCGAGGGAGCGACGCCGCAGGACCTGAACGCGGCCGTCGGAGGTCACCAGGCAGCGATGCAGATGCTGAAGGAGACGATCAACGAGGAGGCGGCCGAGTGGGACCGGGCGGCAGCCGAGCCACGCGGTTCCGACATCGCGGGCACCACCTCGACGCTCCAGGAGCAGATCGCGTGGGCCGCAGCTAAGCGCTGGATCATCCTCGGGCTGAACAAGATCGTCCGGATGTACAGCCCGGTCTCGATCGCTGCTAAGTACGTTGACCGGTTCTTCAAGACCCTGAGGGCCGAGCAGGCGGCCGGACTGGAGGAGATGATGACCGCGTGCCAGGGGGTCGGTGGACCATCGCACAAGGCCGCGCTCTCGCCGCGCACGCTGAACGCGTGGGTGAAGGTCGGATCCGAATTCACGCTGATCCCCATCGCTGTGGGTGGTGCCCTGGCGGGGCTGGTCCTCATCGTCCTCATCGCCTACCTCGTCGGCAGGAAGAGGAGTCACGCAGGCTACCAGACTATCTAGggtacctctagGATCTGCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGGTACCCAGGTGCTGAAGAATTGACCCGGTTCCTCCTGGGCCAGAAAGAAGCAGGCACATCCCCTTCTCTGTGACACACCCTGTCCACGCCCCTGGTTCTTAGTTCCAGCCCCACTCATAGGACACTCATAGCTCAGGAGGGCTCCGCCTTCAATCCCACCCGCTAAAGTACTTGGAGCGGTCTCTCCCTCCCTCATCAGCCCACCAAACCAAACCTAGCCTCCAAGAGTGGGAAGAAATTAAAGCAAGATAGGCTATTAAGTGCAGAGGGAGAGAAAATGCCTCCAACATGTGAGGAAGTAATGAGAGAAATCATAGAATTTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCGGGGGGGGGGGGGCGCTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATCATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTCAGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTGGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTCCCCCCCCCCCCATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGATTGGCTATTGG LAMP-p24CE2 fusion (p24CE2 underlined) encoded by SEQ ID NO: 20SEQ ID NO: 21MAPRSARRPLLLLLLLLLLGLMHCASAAMFMVKNGNGTACIMANFSAAFSVNYDTKSGPKNMTLDLPSDATVVLNRSSCGKENTSDPSLVIAFGRGHTLTLNFTRNATRYSVQLMSFVYNLSDTHLFPNASSKEIKTVESITDIRADIDKKYRCVSGTQVHMNNVTVTLHDATIQAYLSNSSFSRGETRCEQDRPSPTTAPPAPPSPSPSPVPKSPSVDKYNVSGTNGTCLLASMGLQLNLTYERKDNTTVTRLLNINPNKTSASGSCGAHLVTLELHSEGTTVLLFQGGMNASSSRFFLQGIQLNTILPDARDPAFKAANGSLRALQATVGNSYKCNAEEHVRVTKAFSVNIFKNWVQAFKVEGGQFGSVEECLLDENSLEDIVIPMFTALSEGATPQDLNAAVGGHQAAMQMLKETINEEAAEWDRAAAEPRGSDIAGTTSTLQEQIAWAAAKRWIILGLNKIVRMYSPVSIAAKYVDRFFKTLRAEQAAGLEEMMTACQGVGGPSHKAALSPRTLNAWVKVGSEFTLIPIAVGGALAGLVLIVLIAYLVGRKRSHAGYQTI. LAMP-p24CE1 (191H) (gene underlined)SEQ ID NO: 22CCTGGCCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCATGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGATGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGcgggcgcgcgtcgactagcATGGCGCCCCGCAGCGCCCGGCGACCCCTGCTGCTGCTACTGCTGTTGCTGCTGCTCGGCCTCATGCATTGTGCGTCAGCAGCAATGTTTATGGTGAAAAATGGCAACGGGACCGCGTGCATAATGGCCAACTTCTCTGCTGCCTTCTCAGTGAACTACGACACCAAGAGTGGCCCTAAGAACATGACCCTTGACCTGCCATCAGATGCCACAGTGGTGCTCAACCGCAGCTCCTGTGGAAAAGAGAACACTTCTGACCCCAGTCTCGTGATTGCTTTTGGAAGAGGACATACACTCACTCTCAATTTCACGAGAAATGCAACACGTTACAGCGTTCAGCTCATGAGTTTTGTTTATAACTTGTCAGACACACACCTTTTCCCCAATGCGAGCTCCAAAGAAATCAAGACTGTGGAATCTATAACTGACATCAGGGCAGATATAGATAAAAAATACAGATGTGTTAGTGGCACCCAGGTCCACATGAACAACGTGACCGTAACGCTCCATGATGCCACCATCCAGGCGTACCTTTCCAACAGCAGCTTCAGCAGGGGAGAGACACGCTGTGAACAAGACAGGCCTTCCCCAACCACAGCGCCCCCTGCGCCACCCAGCCCCTCGCCCTCACCCGTGCCCAAGAGCCCCTCTGTGGACAAGTACAACGTGAGCGGCACCAACGGGACCTGCCTGCTGGCCAGCATGGGGCTGCAGCTGAACCTCACCTATGAGAGGAAGGACAACACGACGGTGACAAGGCTTCTCAACATCAACCCCAACAAGACCTCGGCCAGCGGGAGCTGCGGCGCCCACCTGGTGACTCTGGAGCTGCACAGCGAGGGCACCACCGTCCTGCTCTTCCAGTTCGGGATGAATGCAAGTTCTAGCCGGTTTTTCCTACAAGGAATCCAGTTGAATACAATTCTTCCTGACGCCAGAGACCCTGCCTTTAAAGCTGCCAACGGCTCCCTGCGAGCGCTGCAGGCCACAGTCGGCAATTCCTACAAGTGCAACGCGGAGGAGCACGTCCGTGTCACGAAGGCGTTTTCAGTCAATATATTCAAAGTGTGGGTCCAGGCTTTCAAGGTGGAAGGTGGCCAGTTTGGCTCTGTGGAGGAGTGTCTGCTGGACGAGAACAGCCTCGAGGATATCGTCATCCCGATGTTCTCGGCGCTCAGCGAGGGAGCGACGCCGCAGGACCTGAACGCGGCCGTCGGAGGTCACCAGGCAGCGATGCAGATGCTGAAGGACACGATCAACGAGGAGGCGGCCGAGTGGGACCGGGCGGCAGCCGAGCCACGCGGTTCCGACATCGCGGGCACCACCTCGACGCTCCAGGAGCAGATCGGGTGGGCCGCAGCTAAGCGCTGGATCATCCTCGGGCTGAACAAGATCGTCCGGATGTACAGCCCGACGTCGATCGCTGCTAAGTACGTTGACCGGTTCTACAAGACCCTGAGGGCCGAGCAGGCGGCCGGACTGGAGGAGATGATGACCGCGTGCCAGGGGGTCGGTGGACCAGGGCACAAGGCCGCGATCTCGCCGCGCACGCTGAACGCGTGGGTGAAGGTCGGATCCGAATTCACGCTGATCCCCATCGCTGTGGGTGGTGCCCTGGCGGGGCTGGTCCTCATCGTCCTCATCGCCTACCTCGTCGGCAGGAAGAGGAGTCACGCAGGCTACCAGACTATCTAGggtacctctagGATCTGCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGGTACCCAGGTGCTGAAGAATTGACCCGGTTCCTCCTGGGCCAGAAAGAAGCAGGCACATCCCCTTCTCTGTGACACACCCTGTCCACGCCCCTGGTTCTTAGTTCCAGCCCCACTCATAGGACACTCATAGCTCAGGAGGGCTCCGCCTTCAATCCCACCCGCTAAAGTACTTGGAGCGGTCTCTCCCTCCCTCATCAGCCCACCAAACCAAACCTAGCCTCCAAGAGTGGGAAGAAATTAAAGCAAGATAGGCTATTAAGTGCAGAGGGAGAGAAAATGCCTCCAACATGTGAGGAAGTAATGAGAGAAATCATAGAATTTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCGGGGGGGGGGGGGCGCTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATCATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTCAGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTGGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTCCCCCCCCCCCCATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGATGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGATTGGCTATTGG LAMP-p24CE1 (p24CE1 underlined) SEQ ID NO: 23MAPRSARRPLLLLLLLLLLGLMHCASAAMFMVKNGNTACIMANFSAAFSVNYDTKSGPKNMTLDLPSDATVVLNRSSCGKENTSDPSLVIAFGRGHTLTLNFTRNATRYSVQLMSFVYNLSDTHLFPNASSKEIKTVESITDIRADIDKKYRCVSGTQVHMNNVTVTLHDATIQAYLSNSSFSRGETRCEQDRPSPTTAPPAPPSPSPSPVPKSPSVDKYNVSGTNGTCLLASMGLQLNLTYERKDNTTVTRLLNINPNKTSASGSCGAHLVTLELHSEDTTVLLFQFGMNASSSRFFLQGIQLNTILPDARDPAFKAANGSLRALQATVGNSYKCNAEEHVRVTKAFSVNIFKVWVQAFKVEGGQFGSVEECLLDENSLEDIVIPMFSALSEGATPQDLNAAVGGHQAAMQMLKDTINEEAAEWDRAAAERPGSDIAGTTSTLQEQIGQAAAKRWIILGLNKIVRMYSPTSIAAKYVDRFYKTLRAEQAAGLEEMMTACQGVGGPGHKAAISPRTLNAWVKVGSEFTLIPIAVGGALAGLVLIVLIAYLVGRKRSHAGYQTI. SP-p24CE2 (235H) (gene underlined)SEQ ID NO: 24CCTGGCCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCATGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGATGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGGcgcgcgtcgacaagaaATGTGGCTCCAGAGCCTGCTACTCCTGGGGACGGTGGCCTGCAGCATCTCGGTCATCCCGATGTTCACGGCGCTCAGCGAGGGAGCGACGCCGCAGGACCTGAACGCGGCCGTCGGAGGTCACCAGGCAGCGATGCAGATGCTGAAGGAGACGATCAACGAGGAGGCGGCCGAGTGGGACCGGGCGGCAGCCGAGCCACGCGGTTCCGACATCGCGGGCACCACCTCGACGCTCCAGGAGCAGATCGCGTGGGCCGCAGCTAAGCGCTGGATCATCCTCGGGCTGAACAAGATCGTCCGGATGTACAGCCCGGTCTCGATCGCTGCTAAGTACGTTGACCGGTTCTTCAAGACCCTGAGGGCCGAGCAGGCGGCCGGACTGGAGGAGATGATGACCGCGTGCCAGGGGGTCGGTGGACCATCGCACAAGGCCGCGCTCTCGCCGCGCACGCTGAACGCGTGGGTGAAGGTCTGATAAgaattcgcggatatcggttaacggatccAGATCTGCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGGTACCCAGGTGCTGAAGAATTGACCCGGTTCCTCCTGGGCCAGAAAGAAGCAGGCACATCCCCTTCTCTGTGACACACCCTGTCCACGCCCCTGGTTCTTAGTTCCAGCCCCACTCATAGGACACTCATAGCTCAGGAGGGCTCCGCCTTCAATCCCACCCGCTAAAGTACTTGGAGCGGTCTCTCCCTCCCTCATCAGCCCACCAAACCAAACCTAGCCTCCAAGAGTGGGAAGAAATTAAAGCAAGATAGGCTATTAAGTGCAGAGGGAGAGAAAATGCCTCCAACATGTGAGGAAGTAATGAGAGAAATCATAGAATTTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCGGGGGGGGGGGGCGCTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATCATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTCAGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTCCCCCCCCCCCCATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGATTGGCTATTGGSP-p24CE2 (p24CE1 underlined) encoded by SEQ ID NO: 24 SEQ ID NO: 25MWLQSLLLLGTVACSISVIPMFTALSEGATPQDLNAAVGGHQAAMQMLKETINEEAAEWDRAAAEPRGSDIAGTTSTLQEQIAWAAAKRWIILGLNKIVRMYSPVSIAAKYVDRFFKTLRAEQAAGLEEMMTACQGVGGPSHKAALSPRTLNAWVKV MCP3-p24CE1 (230H) (gene underlined) SEQ ID NO: 26CCTGGCCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCATGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGATGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGGcgcgcgtcgacaagaaATGTGGAAGCCGATGCCCTCGCCAAGCAACATGAAGGCGTCCGCCGCGCTCCTGTGCCTGCTCCTCACGGCCGCGGCTTTCAGCCCCCAGGGGCTCGCGCAGCCGGTCGGGATCAACACGAGCACGACCTGCTGCTACCGGTTCATCAACAAGAAGATCCCGAAGCAGCGTCTGGAGAGCTACCGCCGGACCACGTCGAGCCACTGCCCGCGGGAGGCGGTCATCTTCAAGACGAAGCTGGACAAGGAGATCTGCGCCGACCCGACGCAGAAGTGGGTTCAGGACTTCATGAAGCACCTGGACAAGAAGACGCAGACGCCGAAGCTGGTCATCCCGATGTTCTCGGCGCTCAGCGAGGGAGCGACGCCGCAGGACCTGAACGCGGCCGTCGGAGGTCACCAGGCAGCGATGCAGATGCTGAAGGACACGATCAACGAGGAGGCGGCCGAGTGGGACCGGGCGGCAGCCGAGCCACGCGGTTCCGACATCGCGGGCACCACCTCGACGCTCCAGGAGCAGATCGGGTGGGCCGCAGCTAAGCGCTGGATCATCCTCGGGCTGAACAAGATCGTCCGGATGTACAGCCCGACGTCGATCGCTGCTAAGTACGTTGACCGGTTCTACAAGACCCTGAGGGCCGAGCAGGCGGCCGGACTGGAGGAGATGATGACCGCGTGCCAGGGGGTCGGTGGACCAGGGCACAAGGCCGCGATCTCGCCGCGCACGCTGAACGCGTGGGTGAAGGTCTGATAAgaattcgcggatatcggttaacggatcaaGATCTGCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGGTACCCAGGTGCTGAAGAATTGACCCGGTTCCTCCTGGGCCAGAAAGAAGCAGGCACATCCCCTTCTCTGTGACACACCCTGTCCACGCCCCTGGTTCTTAGTTCCAGCCCCACTCATAGGACACTCATAGCTCAGGAGGGCTCCGCCTTCAATCCCACCCGCTAAAGTACTTGGAGCGGTCTCTCCCTCCCTCATCAGCCCACCAAACCAAACCTAGCCTCCAAGAGTGGGAAGAAATTAAAGCAAGATAGGCTATTAAGTGCAGAGGGAGAGAAAATGCCTCCAACATGTGAGGAAGTAATGAGAGAAATCATAGAATTTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCGGGGGGGGGGGGCGCTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATCATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTCAGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTCCCCCCCCCCCCATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGATTGGCTATTGGMCP3-p24CE1 (p24CE1 underlined) encoded by SEQ ID NO: 26 SEQ ID NO: 27MWMPMPSPSNMKASAALLCLLLTAAAFSPQGLAQPVGINTSTTCCYRFINKKIPKQRLESYRRTTSSHCPREAVIFKTKLDKEICADPTQKWVQDFMKHLDKKTQTPKLVIPMFSALSEGATPQDLNAAVGGHQAAMQMLKDTINEEAAEWDRAAAEPRGSDIAGTTSTLQEQIGWAAAKRWIILGLNKIVRMYSPTSIAAKYVDRFYKTLRAEQAAGLEEMMTACQGVGGPGHKAAISPRTLNAWVKV. MCP3-p24CE2 (231H) (gene underlined)SEQ ID NO: 28CCTGGCCATTGCATACGTTGTATCCATATCATAATATGACATTTATATTGGCTCATGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGATGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGGcgcgcgtcgacaagaaATGTGGAAGCCGATGCCCTCGCCAAGCAACATGAAGGCGTCCGCCGCGCTCCTGTGCCTGCTCCTCACGGCCGCGGCTTTCAGCCCCCAGGGGCTCGCGCAGCCGGTCGGGATCAACACGAGCACGACCTGCTGCTACCGGTTCATCAACAAGAAGATCCCGAAGCAGCGTCTGGAGAGCTACCGCCGGACCACGTCGAGCCACTGCCCGCGGGAGGCGGTCATCTTCAAGACGAAGCTGGACAAGGAGATCTGCGCCGACCCGACGCAGAAGTGGGTTCAGGACTTCATGAAGCACCTGGACAAGAAGACGCAGACGCCGAAGCTGGTCATCCCGATGTTCACGGCGCTCAGCGAGGGAGCGACGCCGCAGGACCTGAACGCGGCCGTCGGAGGTCACCAGGCAGCGATGCAGATGCTGAAGGAGACGATCAACGAGGAGGCGGCCGAGTGGGACCGGGCGGCAGCCGAGCCACGCGGTTCCGACATCGCGGGCACCACCTCGACGCTCCAGGAGCAGATCGCGTGGGCCGCAGCTAAGCGCTGGATCATCCTCGGGCTGAACAAGATCGTCCGGATGTACAGCCCGGTCTCGATCGCTGCTAAGTACGTTGACCGGTTCTTCAAGACCCTGAGGGCCGAGCAGGCGGCCGGACTGGAGGAGATGATGACCGCGTGCCAGGGGGTCGGTGGACCATCGCACAAGGCCGCGCTCTCGCCGCACGCTGAACGCGTGGGTGAAGGTCTGATAAgaattcgcggatatcggttaacggatccaGATCTGCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGGTACCCAGGTGCTGAAGAATTGACCCGGTTCCTCCTGGGCCAGAAAGAAGCAGGCACATCCCCTTCTCTGTGACACCCTGTCCACGCCCCTGGTTCTTAGTTCCAGCCCCACTCATAGGACACTCATAGCTCAGGAGGGCTCCGCCTTCAATCCCACCCGCTAAAGTACTTGGAGCGGTCTCTCCCTCCCTCATCAGCCCACCAAACCAAACCTAGCCTCCAAGAGTGGGAAGAAATTAAAGCAAGATAGGCTATTAAGTGCAGAGGGAGAGAAAATGCCTCCAACATGTGAGGAAGTAATGAGAGAAATCATAGAATTTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCGGGGGGGGGGGGCGCTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATCATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTCAGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTCCCCCCCCCCCCATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGATTGGCTATTGGMCP3-p24CE2 (p24CE2 is underlined) encoded by SEQ ID NO: 28SEQ ID NO: 29MWKPMPSPSNMKASAALLCLLLTAAAFSPQGLAQPVGINTSTTCCYRFINKKIPKQRLESYRRTTSSHCPREAVIFKTKLDKEICADPTQKWVQDFMKHLDKKTQTPKLVIPMFTALSEGATPQDLNAAVGGHQAAMQMLKETINEEAAEWDRAAAEPRGSDIAGTTSTLQEQIAWAAAKRWIILGLNKIVRMYSPVSIAAKYVDRFFKTLRAEQAAGLEEMMTACQGVGGPSHKAALSPRTLNAWVKV.SP-p24CE1c-alternative conserved element nucleic acid constructSEQ ID NO: 30ATGTGGCTCCAGAGCCTGCTACTCCTGGGGACGGTGGCCTGCAGCATCTCGCAGGGGCAGATGGTCCACCAGGCGATCTCGCCGCGCACGCTGAACGCGTGGGTGAAGGTCCTGGCGAAGGAGGAGAAGGCGTTCAGCCCGGAGGTCATCCCGATGTTCTCGGCGCTCAGCGAGGGAGCGACGCCGCAGGACCTGAACGCGGCCAAGGTCGGAGGTCACCAGGCAGCGATGCAGATGCTGAAGGAGACGATCAACGAGGAGGCGGCCGAGTGGGACCGGGCGGCAGCCGAGCCACGCGGTTCCGACATCGCGGGCACCACCTCGACGCTCCAGGAGCAGATCGGGTGGGCCGCAGCTAAGCGCTGGATCATCCTCGGGCTGAACAAGATCGTCCGGATGTACAGCCCGACGTCGATCGCTGCTAAATACGTTGACCGGTTCTACAAGACCCTGAGGGCCGAGCAGGCGGATTACAAGGACGATGACGACAAGCTGTGATAASP-p24CE1c (p24CE1c underlined) encoded by SEQ ID NO: 30. Includes GM-CSFsignal peptide, CE1 and CE2 replaced by CE8 and CE9, respectively, (relativeto p24 CE “Core1”); lacks CE7, arranged in the configuration of conservedelements: CE 8-9-3-4-5-6 SEQ ID NO: 31MWLQSLLLLGTVACSISQGQMVHQAISPRTLNAWVKKVLAKEEKAFSPEVIPMFSALSEGATPQDLNAAKVGGHQAAMQMLKETINEEAAEWDRAAAEPRGSDIAGTTSTLQEQIGWAAAKRWILGLNKIVRMYSPTSIAAKYVDRFYKTLRAEQADYKDDDDKL conderved element 8 (CE8) SEQ ID NO: 32QGQMVHQAISPRTLNAWVKV conserved element 9 (CE9) SEQ ID NO: 33EEKAFSPEVIPMFSALSEGATPQDLN SP-p24CE2c-alternative SEQ ID NO: 34ATGTGGCTCCAGAGCCTGCTACTCCTGGGGACGGTGGCCTGCAGCATCTCGCAGGGGCAGATGGTCCACCAGGCGCTGTCGCCGCGCACGCTGAACGCGTGGGTGAAGGTCCTGGCGAAGGAGGAGAAGGGGTTCAACCCGGAGGTCATCCCGATGTTCACGGCGCTCAGCGAGGGAGCGACGCCGCAGGACCTGAACGCGGCCAAGGTCGGAGGTCACCAGGCAGCGATGCAGATGCTGAAGGACACGATCAACGAGGAGGCGGCCGAGTGGGACCGGGCGGCAGCCGAGCCACGCGGTTCCGACATCGCGGGCACCACCTCGACGCTCCAGGAGCAGATCGCGTGGGCCGCAGCTAAGCGCTGGATCATCCTCGGGCTGAACAAGATCGTCCGGATGTACAGCCCGGTCTCGATCGCTGCTAAATACGTTGACCGGTTCTTCAAGACCCTGAGGGCCGAGCAGGCGTGATAA SP-p24CE2c (p24CE2c underlined) SEQ ID NO: 35MWLQSLLLLGTVACSISQGQMVHQALSPRTLNAWVKVLAKEEKGFNPEVIPMFTALSEGATPQDLNAAKVGGHQAAMQMLKDTINEEAAEWDRAAAEPRGSDIAGTTSTLQEQIAWAAAKRWIILGLNKIVRMYSPVSIAAKYVDRFFKTLRAEQA SP-p24CE2d alternative nucleic acid conserved element nucleid acidconstruct; in order CE9-3-4-5-6-8 SEQ ID NO: 36ATGTGGCTCCAGAGCCTGCTACTCCTGGGGACGGTGGCCTGCAGCATCTCGGAGGAGAAGGGGTTCAACCCGGAGGTCATCCCGATGTTCACGGCGCTCAGCGAGGGAGCGACGCCGCAGGACCTGAACGCGGCCAAGGTCGGAGGTCACCAGGCAGCGATGCAGATGCTGAAGGACACGATCAACGAGGAGGCGGCCGAGTGGGACCGGGCGGCAGCCGAGCCACGCGGTTCCGACATCGCGGGCACCACCTCGACGCTCCAGGAGCAGATCGCGTGGGCCGCAGCTAAGCGCTGGATCATCCTCGGGCTGAACAAGATCGTCCGGATGTACAGCCCGGTCTCGATCGCTGCTAAATACGTTGACCGGTTCTTCAAGACCCTGAGGGCCGAGCAGGCGGCGCTGCAGGGGCAGATGGTCCACCAGGCGCTGTCGCCGCGCACGCTGAACGCGTGGGTGAAGGTCTGATAA p24CE2d (protein underlined) encoded by SEQ ID NO: 36SEQ ID NO: 37MWLQSLLLGTVACSISEEKGFNPEVIPMFTALSEGATPQDLNAAKVGGHQAAMQMLKDTINEEAAEWDRAAAEPRGSDIAGTTSTLQEQIAWAAAKRWIILGLNKIVRMYSPVSIAAKYVDRFFKTLRAEQAALQGQMVHQALSPRTLNAWVKVSP-24CE1d-conserved element nucleic acid construct; in order CE9-3-4-5-6-8SEQ ID NO: 38ATGTGGCTCCAGAGCCTGCTACTCCTGGGGACGGTGGCCTGCAGCATCTCGGAGGAGAAGGCGTTCAGCCCGGAGGTCATCCCGATGTTCTCGGCGCTCAGCGAGGGAGCGACGCCGCAGGACCTGAACGCGGCCAAGGTCGGAGGTCACCAGGCAGCGATGCAGATGCTGAAGGAGACGATCAACGAGGAGGCGGCCGAGTGGGACCGGGCGGCAGCCGAGCCACGCGGTTCCGACATCGCGGGCACCACCTCGACGCTCCAGGAGCAGATCGGGTGGGCCGCAGCTAAGCGCTGGATCATCCTCGGGCTGAACAAGATCGTCCGGATGTACAGCCCGACGTCGATCGCTGCTAAATACGTTGACCGGTTCTACAAGACCCTGAGGGCCGAGCAGGCGGCGCTGCAGGGGCAGATGGTCCACCAGGCGATCTCGCCGCGCACGCTGAACGCGTGGGTGAAGGTCTGATAA SP-24CE1d encoded by SEQ ID NO: 38 SEQ ID NO: 39MWLQSLLLGTVACSISEEKAFSPEVIPMFSALSEGATPQDLNAAKVGGHQAAMQMLKETINEEAAEWDRAAAEPRGSDIAGTTSTLQEQIGWAAAKRWIILGLNKIVRMYSPTSIAAKYVDRFYKTLRAEQAALQGQMVHQAISPRTLNAWVKVp24CE1d has 6 CE (is identical to p24CE1c except for the CE arrangementwithin the protein) GM-CSF signal peptideCE1 and C2 replaced by CE8 and CE9 respectively, lacks CE7 and has the CEarranged in the configuration CE9-3-4-5-6-8conserved element 8 (CE8-variant for CE2 constructs) SEQ ID NO: 40QGQMVHQALSPRTLNAWVKV conserved element 9 (CE9) SEQ ID NO: 41EEKGFNPEVIPMFTALSEGATPQDLN

Differences between CE for p24 CE polypeptides and variant p24CEpolypeptides is one amino acid per CE except CE9, which differs by 3amino acids

What is claimed is:
 1. A method of inducing an immune response in asubject, the method comprising: (a) administering a first Gag conservedelement nucleic acid that encodes a first Gag conserved elementpolypeptide that comprises six conserved elements from Gag to thesubject, wherein the conserved elements are from different regions ofGag, and further, wherein each conserved element is at least 12 aminoacids in length, but less than 30 amino acids in length and theconserved elements are not contiguous; (b) administering a second Gagconserved element nucleic acid that encodes a second Gag conservedelement polypeptide that comprises at least one variant of a conservedelement polypeptide differs from the variant in the first polypeptide by1, 2, or 3 amino acids; and (c) administering a nucleic acid encoding afull-length Gag protein; wherein the nucleic acid encoding thefull-length Gag protein is administered after the first and the secondGag conserved element nucleic acids, and wherein the region of thenucleic acid encoding the full-length Gag protein encodes only thefull-length Gag protein.
 2. The method of claim 1, wherein the conservedelements are from HIV-1 p24^(gag).
 3. The method of claim 2, wherein thefirst conserved element polypeptide comprises at least one, two, three,four, five, or six conserved element that has an amino acid sequence setforth in SEQ ID NOS:1-7, 32, or
 33. 4. The method of claim 2, whereinthe first conserved element polypeptide comprises conserved elementsthat each have a sequence set forth in SEQ ID NOS:1-7; or the firstconserved element polypeptide comprises conserved elements that eachhave a sequence set forth in SEQ ID NOS:3-6, 32, and
 33. 5. The methodof claim 1, wherein the first conserved element polypeptide comprisesconserved elements that each have a sequence set forth in SEQ ID NOS:1-7and the second conserved element polypeptide comprises conservedelements that each have a sequence set forth in SEQ ID NOS:8-14.
 6. Themethod of claim 1, wherein the conserved element and variant conservedelement each have a sequence set forth in FIG. 1 or in FIG.
 13. 7. Themethod of claim 1, wherein the first Gag conserved element nucleic acidencodes a conserved element polypeptide comprising the sequence of SEQID NO:15 and the second Gag conserved element nucleic acid encodes aconserved element polypeptide comprising the sequence of SEQ ID NO:16.8. The method of claim 1, wherein the first and second Gag conservedelement nucleic acids are administered sequentially.
 9. The method ofclaim 1, wherein the conserved element polypeptides encoded by the firstand second nucleic acid Gag conserved element nucleic acids are eachfused to a GM-CSF signal peptide.
 10. The method of claim 1, wherein thefirst and second nucleic acid Gag conserved element nucleic acids arecontained in the same vector.
 11. The method of claim 1, wherein thefirst and second nucleic acid Gag conserved element nucleic acids arecontained in different vectors.
 12. The method of claim 1, wherein thenucleic acid constructs encoding the conserved element polypeptides andfull-length Gag polypeptide are administered intramuscularly by in vivoelectroporation.
 13. A method of inducing an immune response to an HIVGag protein, the method comprising: (a) administering: (i) a nucleicacid encoding a polypeptide comprising SEQ ID NO:15 and a nucleic acidencoding a polypeptide comprising SEQ ID NO: 16 to a subject; or (ii) anucleic acid encoding a polypeptide comprising p24CE1c as shown in FIG.13 and a nucleic acid encoding a polypeptide comprising p24CE2c as shownin FIG. 13 to the subject; or (iii) a nucleic acid encoding apolypeptide comprising p24CEld as shown in FIG. 13 and a nucleic acidencoding a polypeptide comprising p24CE2d as shown in FIG. 13 to thesubject; and (b) administering a nucleic acid encoding a p55 Gagprotein; wherein the region of the nucleic acid encoding the Gag proteinencodes only the p55 Gag protein.
 14. The method of claim 13, whereinthe polypeptide of SEQ ID NO:15 and the polypeptide of SEQ ID NO:16; orthe polypeptide of p24CE1c and p24CE2c; or the polypeptide of p24CE1dand p24CE2d are encoded by the same vector.
 15. The method of claim 13,wherein the polypeptide of SEQ ID NO:15 and the polypeptide of SEQ IDNO:16; or the polypeptide of p24CE1c and p24CE2c; or the polypeptide ofp24CE1d and p24CE2d are fused to a GM-CSF signal peptide.
 16. The methodof claim 13, wherein the nucleic acids are administered intramuscularlyfollowed by in vivo electroporation.
 17. The method of claim 13, whereinthe nucleic acid encoding p55 gag is administered at least two weeksafter step (a).
 18. A method of inducing an immune response to an HIVgag protein, the method comprising: (a) administering at least onenucleic acid encoding a conserved element polypeptide comprising asequence set forth in SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:21, SE IDNO:23, SEQ ID NO:25, SEQ ID NO:27, SE ID NO:29, SEQ ID NO:31, SEQ IDNO:35, SEQ ID NO:37, or SEQ ID NO:39 to a patient; and (b) administeringa nucleic acid encoding a full-length Gag protein to the patient;wherein the region of the nucleic acid encoding the full-length Gagprotein encodes only the full-length Gag protein.
 19. The method ofclaim 18, wherein the nucleic acid encoding the full-length gag proteinis administered at least 2 weeks after administering the nucleic acidencoding the conserved element polypeptide.